Nadia Canu

Role of the autophagic-lysosomal system on low potassium- induced apoptosis in cultured cerebellar granule cells

Apoptotic and autophagic cell death have been implicated, on the basis of morphological and biochemical criteria, in neuronal loss occurring in neurodegenerative diseases and it has been shown that they may overlap. We have studied the relationship between apoptosis and autophagic cell death in cerebellar granule cells (CGCs) undergoing apoptosis following serum and potassium deprivation. We found that apoptosis is accompanied by an early and marked proliferation of autophagosomal–lysosomal compartments as detected by electron microscopy and immunofluorescence analysis. Autophagy is blocked by hrIGF‐1 and forskolin, two well‐known inhibitors of CGC apoptosis, as well as by adenovirus‐mediated overexpression of Bcl‐2. 3‐Methyladenine (3‐MA) an inhibitor of autophagy, not only arrests this event but it also blocks apoptosis. The neuroprotective effect of 3‐MA is accompanied by block of cytochrome c (cyt c) release in the cytosol and by inhibition of caspase‐3 activation which, in turn, appears to be mediated by cathepsin B, as CA074‐Me, a selective inhibitor of this enzyme, fully blocks the processing of pro‐caspase‐3. Immunofluorescence analysis demonstratesd that cathepsin B, normally confined inside the lysosomal‐endosomal compartment, is released during apoptosis into the cytosol where this enzyme may act as an execution protease. Collectively, these observations indicate that autophagy precedes and is causally connected with the subsequent onset of programmed death.

Isolation and characterization of VGF peptides in rat brain. Role of PC1/3 and PC2 in the maturation of VGF precursor.

The neurotrophin responsive gene vgf is widely expressed in central and peripheral neurones, and in certain neuroendocrine cell populations. Its encoded VGF precursor protein (proVGF1: 617 amino acids in rat, 615 in man, > 85% homology) gives rise to several low molecular weight species. We studied a range of neuroendocrine and neuronal cells, in which VGF‐processing products were prominent with an apparent molecular weight of 20 and 10 kDa (VGF20 and VGF10, respectively). Such peptides were recognized by antibodies specific for the C‐terminal rat VGF nonapeptide, thus indicating that they included the C‐terminus of proVGF. Ectopic expression of the neuroendocrine‐specific prohormone convertases PC1/3 or PC2 in GH3 cells showed that both could generate VGF20, while VGF10 was preferentially produced by PC1/3. Site‐directed mutagenesis was used to identify the KRKRKK488 motif as the target within VGF sequence which leads to the production of VGF20. Molecular characterization of rat VGF10, on the other hand, revealed that this peptide is produced by cleavage at the RPR555 site. By the combined use of high‐resolution separation techniques, matrix‐assisted laser desorption/ionization time of flight (MALDI‐ToF) mass spectrometry and manual Edman degradation we identified in rat brain a VGF fragment analogous to bovine peptide V and two novel peptides also derived from the C‐terminal region of proVGF.

Tissue‐Specific Processing of the Neuroendocrine Protein VGF

Abstract: VGF is a neuroendocrine‐specific gene product that is up‐regulated by nerve growth factor in the PC12 cell line. In rat neuroendocrine tissues two polypeptides of 90 and 80 kDa were detected by an antiserum to an N‐terminal domain of VGF (from residues 4 to 240). In parallel, an antiserum directed against the C‐terminal nonapeptide of VGF (from residues 609 to 617) revealed several additional posttranslational products. Peptides of apparent molecular sizes of 20, 18, and 10 kDa were prominent in nerve tissues and the hypophysis but absent in the adrenal medulla, and their relative abundance varied in distinct regions of the CNS. In PC12 cells VGF was proteolytically processed only after nerve growth factor treatment, and primary cultures of rat cerebellar granule cells accumulated the low‐molecular‐weight forms of VGF during in vitro maturation. In these cells the specific cleavages of VGF occurred in a postendoplasmic reticulum compartment; the processed forms were enriched in the secretory vesicles and were preferentially secreted upon cell membrane depolarization. Distinct differential distribution in the CNS and in vitro release of such posttranslational products indicate that these species may represent biologically relevant forms of VGF that play a role in neuronal communication.

JNK Plays a Key Role in Tau Hyperphosphorylation in Alzheimer's Disease Models

Alzheimers disease (AD) is a major clinical concern, and the search for new molecules to combat disease progression remains important. One of the major hallmarks in AD pathogenesis is the hyperphosphorylation of tau and subsequent formation of neurofibrillary tangles. Several kinases are involved in this process. Amongst them, c-Jun N-terminal kinases (JNKs) are activated in AD brains and are also associated with the development of amyloid plaques. This study was designed to investigate the contribution of JNK in tau hyperphosphorylation and whether it may represent a potential therapeutic target for the fight against AD. The specific inhibition of JNK by the cell permeable peptide D-JNKI-1 led to a reduction of p-tau at S202/T205 and S422, two established target sites of JNK, in rat neuronal cultures and in human fibroblasts cultures. Similarly, D-JNKI-1 reduced p-tau at S202/T205 in an in vivo model of AD (TgCRND8 mice). Our findings support the fundamental role of JNK in the regulation of tau hyperphosphorylation and subsequently in AD pathogenesis.

Identification of a caspase-derived N-terminal tau fragment in cellular and animal Alzheimer's disease models.

Biochemical modifications of tau proteins have been proposed to be among the earliest neurobiological changes in Alzheimers disease (AD) and correlate better with cognitive symptoms than do beta-amyloid plaques. We have recently reported that adenovirus-mediated overexpression of the NH2 26-230aa tau fragment evokes a potent NMDA-mediated neurotoxic effect in primary neuronal cultures. In order to assess whether such N-terminal tau fragment(s) are indeed produced during apoptosis or neurodegeneration in vivo, we attempted to ascertain their presence in cell and animal models using an anti-tau antibody directed against the N-terminal sequence of human protein located downstream of the caspase(s)-cleavage site DRKD(25)-QGGYTMHQDQ. We provide biochemical evidence that a caspase(s)-cleaved NH2-terminal tau fragment of 20-22 kDa, consistent with the size of the NH2 26-230aa neurotoxic fragment of tau, is generated in vitro in differentiated human SH-SY5Y cells undergoing apoptosis by BDNF withdrawal or following treatment with staurosporine. In addition this NH2-terminally cleaved tau fragment, whose expression correlates with a significant up-regulation of caspase(s) activity, is also specifically detected in vivo in the hippocampus of 15 month-old AD11 transgenic mice, a model in which a progressive AD-like neurodegeneration is induced by the expression of transgenic anti-NGF antibodies. The results support the idea that aberrant activation of caspase(s), following apoptotic stimuli or neurodegeneration insults, may produce one or more toxic NH2 tau fragments, that further contribute to propagate and increase cellular dysfunctions in AD.

In vitro cultured neurons for molecular studies correlating apoptosis with events related to Alzheimer disease.

This short review analyses the possible molecular events linking a general program of death such as apoptosis to highly specific intracellular pathways involving the function and degradation of two proteins tau and amyloid precursor protein which in their aggregated state constitute the hallmark of Alzheimer disease. By surveying the recent studies carried out in ‘in vitro’ neuronal cultures with special emphasis to cerebellar granule neurons the apparent correlation between onset of apoptosis, tau cleavage with formation of potential toxic fragments, and activation of an amyloidogenic route are discussed. Within this framework, proteasomes seem to play a crucial role upstream of the proteolytic cascade involving calpain(s) and caspase(s) by contributing to tau and amyloid precursor protein-altered breakdown and consequent tendency to aggregation of their degradation fragments. Thus, apoptotic death due to altered supply of anti apoptotic agents, neurotrophic factors, deafferentiation or other causes, may constitute a major trigger of the onset of Alzheimer disease.

Increased levels of p70S6 phosphorylation in the G93A mouse model of Amyotrophic Lateral Sclerosis and in valine-exposed cortical neurons in culture

The higher risk factor for Amyotrophic Lateral Sclerosis (ALS) among Italian soccer players is a question that is still debated. One of the hypotheses that have been formulated to explain a possible link between ALS and soccer players is related to the abuse of dietary supplements and drugs for enhancing sporting performance. In particular, it has been reported that branched-chain amino acids (BCAAs) are widely used among athletes as nutritional supplements. To observe the possible effect of BCAAs on neuronal electrical properties, we performed electrophysiological experiments on Control cultured cortical neurons and on neurons after BCAA treatment. BCAA-treated neurons showed hyperexcitability and rapamycin was able to suppress it and significantly reduce the level of mTOR, Akt and p70S6 phosphorylation. Interestingly, the hyperexcitability previously reported in cortical neurons from a genetic mouse model of ALS (G93A) was also reversed by rapamycin treatment. Moreover, both G93A and valine-treated neurons presented significantly higher levels of Pp70S6 when compared to control neurons, strongly indicating the involvement of this substrate in ALS pathology. Finally, we performed electrophysiological experiments on motor cortex slices from Control and G93A mice and those fed with a BCAA-enriched diet. We observed that neuron excitability was comparable between G93A and BCAA-enriched diet mice, but was significantly higher than in Control mice. These findings, besides strongly indicating that BCAAs specifically induce hyperexcitability, seem to suggest the involvement of p70S6 substrate in ALS pathology.

Substance P provides neuroprotection in cerebellar granule cells through Akt and MAPK/Erk activation: Evidence for the involvement of the delayed rectifier potassium current

In the current study, we have evaluated the ability of substance P (SP) and other neurokinin 1 receptor (NK1) agonists to protect, in a dose- and time-dependent manner, primary cultures of rat cerebellar granule cells (CGCs) from serum and potassium deprivation-induced cell death (S-K5). We also established the presence of SP high affinity NK1 transcripts and the NK1 protein localization in the membrane of a sub-population of CGCs. Moreover, SP significantly and dose-dependently reduced the Akt 1/2 and Erk1/2 dephosphorylation induced by S-K5 conditions, as demonstrated by Western blot analysis. Surprisingly, in SP-treated CGCs caspase-3 activity was not inhibited, while the calpain-1 activity was moderately reduced. Corroborating this result, SP blocked calpain-mediated cleavage of tau protein, as demonstrated by the reduced appearance of a diagnostic fragment of 17 kDa by Western blot analysis. In addition, SP induced a significant reduction of the delayed rectifier K+ currents (Ik) in about 42% of the patched neurons, when these were evoked with depolarizing potential steps. Taken together, the present results demonstrate that the activation of NK1 receptors expressed in CGCs promote the neuronal survival via pathways involving Akt and Erk activation and by inhibition of Ik which can contribute to the neuroprotective effect of the peptide.

Interaction of Tau with Fe65 links tau to APP

The beta-amyloid precursor protein APP and the microtubule-associated protein Tau play a crucial role in the pathogenesis of Alzheimers disease (AD). However, the possible molecular events linking these two proteins are still unknown. Here, we show that Fe65, one of the ligands of the APP cytodomain, is associated with Tau in vivo and in vitro, as demonstrated by co-immunoprecipitation, co-localization, and FRET experiments. Deletion studies indicated that the N-terminal domain of Tau and the PTB1 domain of Fe65 are required for this association. This interaction is regulated by the phosphorylation of Tau at selected sites, by glycogen synthase kinase-3beta (GSK3beta) and cyclin-dependent kinase 5 (Cdk5), and requires an intact microtubule network. Furthermore, laser scanner microscopy and co-immunoprecipitation experiments provide preliminary evidence of possible complex(es) involving Tau, Fe65, APP. These findings open new perspectives for the study of the possible crosstalk between these proteins in the pathogenesis of AD.

PC4 Coactivates MyoD by Relieving the Histone Deacetylase 4-Mediated Inhibition of Myocyte Enhancer Factor 2C

ABSTRACT 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.