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APECED (Autoimmune Polyendocrinopathy-Candidiasis-Ectodermal Distrophy) is a rare autosomal recessive disorder. We previously found that sera samples from 9/14 patients revealed autoantibodies (Auto-Abs) reacting with cerebellum (GABAergic cells, n=5) and substantia nigra (SN; dopaminergic cells, n=5) [1]. Follow-up of the large majority of these patients was perfomed at least 10 years after the previous investigation. Indeed, on these patients, and on control age-matched subjects (n=14), we performed brain examinations using an MRI scanner. Obtained images were used to evaluate the volumes of white and gray matter (W.M and G.M., respectively) as well as the ventricles (III and IV). In addition, we used immunohistochemistry (IHC) on tissues from rat brain (after perfusion with 4% paraformaldehyde) in order to confirm the previous immunoreactivities or found new Auto-Abs cell targets. The brain MR revealed a reduction of G.M (p = 0.042) and cerebellum (p = 0.0012), and an increase of ventricles (p = 0.0001), compared to controls. Through IHC, after 10 years, we found 11/14 patients producing Auto-Abs against different brain neuronal cells. In detail, among the patients previously investigated and containing Auto-Abs against GABAergic perikarya in the cerebellum, 3 still contained the same immunoreactivity while 1 was unavailable, and 1 lost the reactivity. Instead, as to Auto-Abs against dopaminergic perikarya in the SN, 4 patients confirmed their previous reactivity, while 3 previously negative patients, revealed novel positivity (in total, n=7). A new immunoreactivity against the 5HT cells in the brainstem were also revealed in the same patients with Auto-Abs to SN (n=7). In conclusion, the co-presence of brain volume changes and neuronal Auto-Abs in APECED patients could suggest an autoimmune manifestation at the brain level that should be taken in consideration., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

TLQP peptides (TLQPp) act on synaptic strengthening or against neuronal apoptosis but their involvement in Amyotrophic Lateral Sclerosis (ALS) is not well known. We used an ALS animal model (SOD1-G93A) and human tissues (plasma and fibroblasts from both patients and controls), to address the TLQPp expression and their modulation in ALS mechanisms. Mouse motor neuronal cells (NSC-34), were used to study the neuroprotective role of the TLQP-21 against oxidative stress induced by Sodium Arsenite. TLQPp were immunoquantified by ELISA, their cell expression studied by immunofluorescence while cell viability was addressed by MTT assay. In the NSC-34 naïve cells, TLQPp were found within the cytoplasm, axons and growth cones. Upon stress, they appeared immunolocalized exclusively within a cytoplasmatic area, close to the nucleus, (probably the Golgi). Moreover, under stress, TLQPp levels were reduced (42%, p=3.1x10-6), while the exogenous increase of TLQP-21 improved cell viability (13%, p= 0.018). In naïve fibroblasts, TLQPp were also similarly localized in an area, likely the Golgi, of patients and controls while their reduction (31%, p=0.03) was observed in cells with TARDBP-A382T mutation. Stress granules only appeared after SA treatment, and did not contain TLQPp. In plasma, a reduced release of TLQPp was associated with the beginning of the clinical motor symptoms in patients (12%, p=0.026), and with a pre-symptomatic stage in mutant mice (26.3%, p=0.048). Finally, in mouse spinal cord we identified by vescicular acetylcholine transporter (VAChT) antibody, a specific localization of the TLQPp in the motor neurons with a reduction in the pre-symptomatic stage of mutant mice. In conclusion, TLQPp are reduced in the stressed NSC-34 cells, mutant mouse motor neurons as well as in fibroblasts with TARDBP-A382T mutation that also induces production of reactive oxygen specie. Hence, we suggest that their reduction may occur in response to oxidative stress. They could be also considered as early biomarkers, while TLQP-21 acts as neuroprotective factor., Italian Journal of Anatomy and Embryology, Vol. 122, No. 1 (Supplement) 2017

Parkinson’s disease (PD) is a neurological disorder characterized by a progressive degeneration of dopaminergic neurons of the substantia nigra, that results in dopamine depletion in the striatum with a deregulation of a number of substances/ neurotrasmitters. A previous study demonstrated a clear-cut decrease of certain peptides derived from the VGF gene in the parietal cortex from patients affected by PD. Hence, we used an animal model of PD, and plasma samples from PD patients and control subjects to investigate the role of VGF in PD. Rats (Sprague Dawley) were treated with injections of 6 hydroxydopamine into the medial forebrain bundle, anesthetized and perfused with 4% paraformaldehyde 3 weeks (group 1: n= 8) or 6 weeks (group 2: n=17) after injection. In addition, subcutaneous injections of L-DOPA (6mg/ Kg) were carried out one week before the sacrifice in rats from group 1 and 2 (n=4 and n=6, respectively). We raised antibodies to the C-terminal end of the human VGF sequence, for immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA, IC50 = 15 pmol/ml). For pilot experiments, human plasma samples were taken from subjects affected by PD (n=13, age range: 51–84 years), and from age matched controls (n=10), to be used for ELISA experiments. We analysed the substantia nigra (SN) of the PD rats using VGF-C-terminus antiserum in double staining with antibodies to glutamate decarboxylase (GAD), tyrosine hydroxylase (TH), serotonin and Substance P. In the control side, VGF-C-terminus peptides were found in neurone terminals/axons within the pars compacta (SNC) and reticulata (SNR) of the substantia nigra, co-localised with GAD and substance P (but not with serotonin or TH) and in closed connection with TH cell bodies of the SNC. In the PD side, while the GAD staining remains unaffected, VGF-C-terminus and substance P immnureactivities disappeared (compared to the control side) in a large population of neurone terminals of both SNC and SNR, and in relation with the TH decrease. When L-DOPA was applied to the animals, VGF-C-terminus immunoreactivity on the PD side returned comparable to the control side. By ELISA, high concentrations of proVGF, in the order of 600-2000 pmol/ml were shown in the plasma from controls with a significant decrease in PD samples (p=0,0029). In conclusion, using the animal models, we showed here for the first time that VGF is involved into nigro-striatal circuits and down-regulated in PD. Besides, of a great interest, is the finding that a deregulation of VGF can occur also in humans., Italian Journal of Anatomy and Embryology, Vol 119, No 1 (Supplement) 2014

vgf is one of the few genes selectively induced in the hypothalamus of Siberian hamsters upon their typical change from an obese phenotype (long day adaptation, during summer) to a lean, catabolic phenotype (short day, or winter adaptation). In fact, the i.c.v. injection of the VGF-derived peptide TLQP-21 caused hypophagia and a decrease in body weight in long day hamster. Hence, we studied VGF multi-peptide profiles in brain cortex and hypothalamus of (male) Siberian hamsters, in the long day (LD) versus short day (SD) adapted state. Specific antisera were produced against short sequences at the C- or N-termini of VGF, and of several known/predicted VGF-derived products: TLQP, NERP-1, and PGH peptides, and used in immunohistochemistry (IHC) and ELISA. Hamsters were perfused with 4% paraformaldehyde (n= 4/group) for IHC or used for tissue sampling and extraction (n= 7/group). In IHC, VGF C- and N- terminus peptides were brightly labelled, as well as most abundant. They were found in both perikarya and axons, in different layers of brain cortex and in multiple hypothalamic areas, including the paraventricular (PVN), suprachiasmatic (SCN), supraoptic (SON) and arcuate nuclei, the lateral and anterior hypothalamic areas, and the median eminence (ME). TLQP peptides were largely restricted to SCN perikarya, and ME axons, while PGH and NERP-1 peptides were revealed in perikarya of the brain cortex, in ME axons, and certain perikarya of PVN and SON (NERP-1 only). Most VGF peptides studied were well represented in tissue extracts of hypothalamus and cortex, VGF C- and N- terminus peptides being again most abundant (hypothalamus: 1.8±0.3 and 10.9±0.6; cortex: 0.7±0.1 and 5±0.3 nmol/g, mean±SEM, C- and N-terminus, respectively, LD animals). A selective decrease in certain VGF peptides was revealed in SD animals, compared to LD ones, so that NERP-1 peptides were decreased in hypothalamus and cortex (61.3±12.7% and 45.8±11.1% of LD animals, respectively, mean±SEM, p, Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

The vgf gene shows delayed-early induction by neuronal Growth Factors. It encodes the VGF protein precursor (615/617 aa, in man/rat), which yields an incompletely characterized neuropeptides, some of which are involved in synaptic strengthening, anti-depressant and anti-apoptotic actions. Proteomic studies revealed clear-cut reduction of a 4.8kD VGF fragment in human CS fluid from ALS patients. In primary cultures of spinal motoneurons from SOD1-G93A mice (as a model of ALS), exogenous adenoviral expression of VGF attenuated excitotoxic injury. In the same animals, a decrease of VGF was seen in CSF and serum, in parallel to progression of muscle weakness. Hence, we aimed at delineating VGF multi-peptide profiles in (normal) rat motoneurons, as well as possible changes of such peptides in plasma from human ALS patients vs controls. Specific antisera were raised against short sequences at the C- and N-termini (“C-t”, and “N-t”) of the VGF precursor, and of known / predicted cleaved VGF peptides, and were used in immunohistochemistry and ELISA. Spinal cord, frontal cortex and brainstem samples were taken for immunohistochemistry from rats (N=9) perfused with 4% paraformaldehyde. Plasma was obtained from ALS and control subjects (N=26/14 respectively, age range: 50-60yrs). Immunohistochemistry showed selective localization of VGF peptides in motoneurons in all regions tested. In the frontal cortex, VGF C-t peptides were widely distributed in perikarya of internal layers (III-VI), and in axons in superficial layers (I-II). Axons and terminal were brightly stained in the ventral horn of the spinal cord, and in a number of brainstem motor nuclei (trigeminal, facial, hypoglossal and ambigus N.). Conversely, VGF N-t antibodies labelled mostly perikarya in all areas tested, while NERP-1 and –2, and PGH (VGF422-430) peptides were restricted to occasional cell bodies in frontal cortex. In a few sections, PGH and TLQP-21(VGF556-576) labelling was seen in ventral horn perikarya. As to human plasma, NERP-1 and –2 as well as PGH peptides showed a significant reduction in ALS patients, versus controls (peptide concentration measured: 37%, 43% and 29% of controls, respectively, p, Italian Journal of Anatomy and Embryology, Vol 116, No 1 (Supplement) 2011

Colorectal cancer (CRC) is the fourth cause of death from cancer worldwide mainly due to the high incidence of drug-resistance. During a screen for new actionable targets in drug-resistant tumours we recently identified p65BTK–a novel oncogenic isoform of Bruton's tyrosine kinase. Studying three different cohorts of patients here we show that p65BTK expression correlates with histotype and cancer progression. Using drug-resistant TP53-null colon cancer cells as a model we demonstrated that p65BTK silencing or chemical inhibition overcame the 5-fluorouracil resistance of CRC cell lines and patient-derived organoids and significantly reduced the growth of xenografted tumours. Mechanistically, we show that blocking p65BTK in drug-resistant cells abolished a 5-FU-elicited TGFB1 protective response and triggered E2F-dependent apoptosis. Taken together, our data demonstrated that targeting p65BTK restores the apoptotic response to chemotherapy of drug-resistant CRCs and gives a proof-of-concept for suggesting the use of BTK inhibitors in combination with 5-FU as a novel therapeutic approach in CRC patients. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Bruton's tyrosine kinase (BTK) is essential for B-cell proliferation/differentiation and it is generally believed that its expression and function are limited to bone marrow-derived cells. Here, we report the identification and characterization of p65BTK, a novel isoform abundantly expressed in colon carcinoma cell lines and tumour tissue samples. p65BTK protein is expressed, through heterogeneous nuclear ribonucleoprotein K (hnRNPK)-dependent and internal ribosome entry site-driven translation, from a transcript containing an alternative first exon in the 5'-untranslated region, and is post-transcriptionally regulated, via hnRNPK, by the mitogen-activated protein kinase (MAPK) pathway. p65BTK is endowed with strong transforming activity that depends on active signal-regulated protein kinases-1/2 (ERK1/2) and its inhibition abolishes RAS transforming activity. Accordingly, p65BTK overexpression in colon cancer tissues correlates with ERK1/2 activation. Moreover, p65BTK inhibition affects growth and survival of colon cancer cells. Our data reveal that BTK, via p65BTK expression, is a novel and powerful oncogene acting downstream of the RAS/MAPK pathway and suggest that its targeting may be a promising therapeutic approach.Oncogene advance online publication, 25 January 2016; doi:10.1038/onc.2015.504.