Nature-Inspired Multifunctional Ligands: Focusing on Amyloid-Based Molecular Mechanisms of Alzheimer's Disease.

The amyloidogenic pathway is a prominent feature of Alzheimer's disease (AD). However, growing evidence suggests that a linear disease model based on ß-amyloid peptide (Aß) alone is not likely to be realistic, which therefore calls for further investigations on the other actors involved in the play. The pro-oxidant environment induced by Aß in AD pathology is well established, and a correlation among Aß, oxidative stress, and conformational changes in p53 has been suggested. In this study, we applied a multifunctional approach to identify allyl thioesters of variously substituted trans-cinnamic acids for which the pharmacological profile was strategically tuned by hydroxy substituents on the aromatic moiety. Indeed, only catechol derivative 3 [(S)-allyl (E)-3-(3,4-dihydroxyphenyl)prop-2-enethioate] inhibited Aß fibrilization. Conversely, albeit to different extents, all compounds were able to decrease the formation of reactive oxygen species in SH-SY5Y neuroblastoma cells and to prevent alterations in the conformation of p53 and its activity mediated by soluble sub-lethal concentrations of Aß. This may support an involvement of oxidative stress in Aß function, with p53 emerging as a potential mediator of their functional interplay.

Defective DNA repair and increased chromatin binding of DNA repair factors in Down syndrome fibroblasts.

Down syndrome (DS) is characterized by genetic instability, neurodegeneration, and premature aging. However, the molecular mechanisms leading to this phenotype are not yet well understood. Here, we report that DS fibroblasts from both fetal and adult donors show the presence of oxidative DNA base damage, such as dihydro-8-oxoguanine (8-oxodG), and activation of a DNA damage response (DDR), already during unperturbed growth conditions. DDR with checkpoint activation was indicated by histone H2AX and Chk2 protein phosphorylation, and by increased p53 protein levels. In addition, both fetal and adult DS fibroblasts were more sensitive to oxidative DNA damage induced by potassium bromate, and were defective in the removal of 8-oxodG, as compared with age-matched cells from control healthy donors. The analysis of core proteins participating in base excision repair (BER), such as XRCC1 and DNA polymerase ß, showed that higher amounts of these factors were bound to chromatin in DS than in control cells, even in the absence of DNA damage. These findings occurred in concomitance with increased levels of phosphorylated XRCC1 detected in DS cells. These results indicate that DS cells exhibit a BER deficiency, which is associated with prolonged chromatin association of core BER factors.

CBP and p300 acetylate PCNA to link its degradation with nucleotide excision repair synthesis.

The proliferating cell nuclear antigen (PCNA) protein serves as a molecular platform recruiting and coordinating the activity of factors involved in multiple deoxyribonucleic acid (DNA) transactions. To avoid dangerous genome instability, it is necessary to prevent excessive retention of PCNA on chromatin. Although PCNA functions during DNA replication appear to be regulated by different post-translational modifications, the mechanism regulating PCNA removal and degradation after nucleotide excision repair (NER) is unknown. Here we report that CREB-binding protein (CBP), and less efficiently p300, acetylated PCNA at lysine (Lys) residues Lys13,14,77 and 80, to promote removal of chromatin-bound PCNA and its degradation during NER. Mutation of these residues resulted in impaired DNA replication and repair, enhanced the sensitivity to ultraviolet radiation, and prevented proteolytic degradation of PCNA after DNA damage. Depletion of both CBP and p300, or failure to load PCNA on DNA in NER deficient cells, prevented PCNA acetylation and degradation, while proteasome inhibition resulted in accumulation of acetylated PCNA. These results define a CBP and p300-dependent mechanism for PCNA acetylation after DNA damage, linking DNA repair synthesis with removal of chromatin-bound PCNA and its degradation, to ensure genome stability.

Zyxin is a novel target for ß-amyloid peptide: characterization of its role in Alzheimer's pathogenesis.

Zyxin is an adaptor protein recently identified as a novel regulator of the homeodomain-interacting protein kinase 2 (HIPK2)-p53 signaling in response to DNA damage. We recently reported an altered conformational state of p53 in tissues from patients with Alzheimer 's disease (AD), because of a deregulation of HIPK2 activity, leading to an impaired and dysfunctional response to stressors. Here, we examined the molecular mechanisms underlying the deregulation of HIPK2 activity in two cellular models, HEK-293 cells and SH-SY5Y neuroblastoma cells differentiated with retinoic acid over-expressing the amyloid precursor protein, focusing on the evidence that zyxin expression is important to maintain HIPK2 protein stability. We demonstrated that both beta-amyloid (Aß) 1-40 and 1-42 induce zyxin deregulation, thus affecting the transcriptional repressor activity of HIPK2 onto its target promoter, metallothionein 2A, which is in turn responsible for the induction of an altered conformational state of p53. We demonstrate for the first time that zyxin is a novel target of Aß activities in AD. These results may help the studies on the pathogenesis of AD, through the fine dissection of events related to beta-amyloid activities.

p300/CBP acetyl transferases interact with and acetylate the nucleotide excision repair factor XPG.

Nucleotide excision repair (NER) is an important DNA repair mechanism through which cells remove bulky DNA lesions. Following DNA damage, the histone acetyltransferase (HAT) p300 (also referred to as lysine acetyltransferase or KAT) is known to associate with proliferating cell nuclear antigen (PCNA), a master regulator of DNA replication and repair processes. This interaction, which results in HAT inhibition, may be dissociated by the cell cycle inhibitor p21(CDKN1A), thereby restoring p300 activity; however, the role of this protein interplay is still unclear. Here, we report that silencing p300 or its homolog CREB-binding protein (CBP) by RNA interference (RNAi) significantly reduces DNA repair synthesis in human fibroblasts. In addition, we determined whether p300 and CBP may associate with and acetylate specific NER factors such as XPG, the 3'-endonuclease that is involved in the incision/excision step and is known to interact with PCNA. Our results show that p300 and CBP interact with XPG, which has been found to be acetylated in vivo. XPG is acetylated by p300 in vitro, and this reaction is inhibited by PCNA. Knocking down both p300/CBP by RNAi or by chemical inhibition with curcumin greatly reduced XPG acetylation, and a concomitant accumulation of the protein at DNA damage sites was observed. The ability of p21 to bind PCNA was found to regulate the interaction between p300 and XPG, and an abnormal accumulation of XPG at DNA damage sites was also found in p21(-/-) fibroblasts. These results indicate an additional function of p300/CBP in NER through the acetylation of XPG protein in a PCNA-p21 dependent manner.

Dysfunction of the ubiquitin-proteasome system in the cerebellum of aging Ts65Dn mice.

In the cerebellum of adult-aging Ts65Dn mice, a murine model of Down syndrome, Purkinje cells undergo degeneration. Searching for the cause of Purkinje cell degeneration, we have studied the ubiquitin-proteasome system (UPS) in the cerebellum of aging Ts65Dn mice. Inhibition of UPS is sufficient to induce neuron degeneration and death. Proteasome chymotrypsin-like proteolytic activity was reduced by 35% in the cerebellum of Ts65Dn mice in comparison with euploid animals. Accordingly, Western blot analysis of ubiquitin showed an increase in ubiquitinated proteins. Immunocytochemistry for ubiquitin revealed strongly positive intranuclear inclusions in Purkinje cells and large neurons of cerebellar nuclei. The Western blot analysis of ubiquitin in nuclear protein extracts confirmed the increase of ubiquitinated proteins in the cell nuclei. After FUS immunocytochemistry, large intranuclear inclusions were visible in Purkinje cells and large neurons of cerebellar nuclei in Ts65Dn mice. Together, data indicate a possible role for proteasome inhibition in the cerebellar neurodegeneration in Ts65Dn mice.

A single episode of neonatal seizures alters the cerebellum of immature rats.

PURPOSE: to test whether a single episode of early-life seizures may interfere with the development of the cerebellum. The cerebellum is particularly vulnerable in infants, since it is characterized by an important postnatal histogenesis that leads to the settling of adult circuitry. METHODS: seizures were induced in 10-day-old Wistar rats with a single convulsive dose (80µg/g b.w., s.c.) of pentylentetrazole (PTZ). Immediately after rats were treated with (3)H-thymidine ((3)HTdR, 2.5µCi/g b.w, s.c.). Rats were killed 4h later and paraffin sections of the cerebellar vermis were processed for (3)HTdR autoradiography and immunocytochemistry for 2/3 subunits of AMPA glutamate receptor (GluR2/3), glutamate transporter 1 (GLT1) and calbindin. RESULTS: seizures reduced the proliferation rate of cells in the external germinal layer. Purkinje cells showed increased GluR2/3 immunoreactivity. However, some Purkinje cells were unstained or lost. Increased GLT1 immunoreactivity was present in glial cells surrounding Purkinje cells. Calbindin immunoreaction confirmed that some Purkinje cells were missed. The remaining Purkinje cells showed large spheroids along the course of their axon. CONCLUSIONS: data indicate that seizures lead to a loss and alteration of Purkinje cells in the cerebellum of immature rats. Since at 10 days of life Purkinje cells are no more proliferating, the loss of Purkinje cells should be permanent.

Beta-amyloid overload does not directly correlate with SAPK/JNK activation and tau protein phosphorylation in the cerebellar cortex of Ts65Dn mice.

It is known that in the nervous tissue beta-amyloid overproduction and its extracellular or intracellular deposition can activate mitogen-activated protein kinases involved in tau protein phosphorylation. Hyperphosphorylated tau is not more able to bind neuron microtubules, leading to their disassembly and axon degeneration. We have previously described that at 10 months of age in the cerebellum of Ts65Dn mice, which are partially trisomic for the chromosome 16 and are considered a valuable model for Down syndrome, Purkinje cells undergo axon degeneration. Taking into consideration that Ts65Dn mice carry three copies of the gene encoding for the amyloid precursor protein, to characterize potential signaling events triggering the degenerative phenomenon, specific antibodies were used to examine the role of beta-amyloid overload in the activation of the stress activated kinase/c-jun N-terminal kinase (SAPK/JNK) and tau protein phosphorylation in the cerebellar cortex of 12-month-old Ts65Dn mice. We found small extracellular deposits of beta-amyloid at the borderline between the granule cell layer and the white matter, i.e., in the vicinity of the area where calbindin immunostaining of Purkinje cell axons revealed clusters of newly formed terminals of injured axons. Moreover, intracellular deposits were present in the somata of Purkinje cells. The level of activation of SAPK/JNK was greatly increased. The activation occurred in the "pinceaux" made by basket interneuron axons at the axon hillock of Purkinje cells. Antibody directed against tau protein phosphorylated at Ser-396/Ser-404 revealed positive NG2 cells and Bergman fibers in the molecular layer and oligodendrocytes in the white matter. Data indicate that beta-amyloid extracellular deposits could have exerted a local cytotoxic effect, leading to Purkinje cell axon degeneration. The activation of SAPK/JNK in basket cell "pinceaux" may be a consequence of altered functionality of Purkinje cells and may represent an attempt of basket cells of synaptic remodeling. Moreover, the findings for tau protein phosphorylation suggest that Ts65Dn mice are affected by a cerebellar glial tauopathy.

Degradation of p21CDKN1A after DNA damage is independent of type of lesion, and is not required for DNA repair.

The inhibitor of cyclin-dependent kinases p21CDKN1A plays a fundamental role in several pathways involved in the DNA damage response, like checkpoint-mediated cell cycle arrest, transcription, apoptosis, and DNA repair. Although p21 protein level is regulated by proteasomal degradation, the relationship of this process with DNA repair pathways is not yet clear. In addition, the role of protein/protein interaction in regulating turnover of p21 protein, is controversial. Here, we show that in human fibroblasts treated with agents inducing lesions repaired through nucleotide excision repair (NER), or base excision repair (BER), p21 degradation was triggered more by the extent, than by the type of DNA damage, or consequent DNA repair pathway. In fact, lowering the amount of DNA damage resulted in an increased stability of p21 protein. Overexpression of p21 was found to obscure degradation, both for p21wt and a p21 mutant unable to bind PCNA (p21PCNA-). However, when expressed to lower levels, turnover of p21 protein after DNA damage was greatly influenced by interaction with PCNA, since p21PCNA- was more efficiently degraded than wild-type protein. Interestingly, a p21 mutant protein unable to localize in the nucleus because of mutations in the NLS region, was not degraded after DNA damage, thus indicating that nuclear localization is necessary for p21 turnover. Removal of p21 was not required for NER activity, since inhibition of p21 degradation by caffeine did not affect the UV-induced recruitment of repair proteins, such as PCNA and DNA polymerase delta, nor significantly influence DNA repair synthesis, as determined by autoradiography. These results indicate that degradation of p21 is not dependent on a particular DNA repair pathway, and is not required for efficient DNA repair.

Axonal abnormalities in cerebellar Purkinje cells of the Ts65Dn mouse.

Ts65Dn mice are a genetic model for Down syndrome. Among others, these mice have cerebellar pathology features which parallel those seen in Down syndrome patients. Both individuals with Down syndrome and Ts65Dn mice have reduced cerebellar volume and numbers of granule and Purkinje cells. In this report, we describe morphological abnormalities of axons of Purkinje cells in the cerebellum of Ts65Dn mice, by using anti-calbindin immunocytochemistry. A consistent number of Purkinje cells shows axons bearing giant varicosities along their transit through the granular layer. The cerebellar arbor vitae made by fasciculated Purkinje cell axons has a patchy appearance, some tracks being devoid of calbindin staining. The infraganglionic plexus, formed by recurrent collaterals of Purkinje cell axons, has enormously increased density, which is evidence for a compensatory reaction to degeneration of distal segments of axons. These alterations are accompanied by strong glial reaction as evidenced by GFAP immunocytochemistry. Moreover, the alterations are more consistent in the anterior lobules of the vermis and intermediate cortex. The axonal pathology of Purkinje cells may explain the impairment in cerebellar functions observed in Ts65Dn mice at the adulthood.

Altered neuron excitability and synaptic plasticity in the cerebellar granular layer of juvenile prion protein knock-out mice with impaired motor control.

Although the role of abnormal prion protein (PrP) conformation in generating infectious brain diseases (transmissible spongiform encephalopathy) has been recognized, the function of PrP in the normal brain remains mostly unknown. In this investigation, we considered the effect of PrP gene knock-out (PrP(0/0)) on cerebellar neural circuits and in particular on granule cells, which show intense PrP expression during development and selective affinity for PrP. At the third postnatal week, when PrP expression would normally attain mature levels, PrP(0/0) mice showed low performance in the accelerating rotarod and runway tests and the functioning of 40% of granule cells was abnormal. Spikes were slow, nonovershooting, and nonrepetitive in relation with a reduction in transient inward and outward membrane currents, and also the EPSPs and EPSCs had slow kinetics. Overall, these alterations closely resembled an immature phenotype. Moreover, in slow-spiking PrP(0/0) granule cells, theta-burst stimulation was unable to induce any long-term potentiation. This profound impairment in synaptic excitation and plasticity was associated with a protracted proliferation of granule cells and disappeared at P40-P50 along with the recovery of normal motor behavior (Büeler et al., 1992). These results suggest that PrP plays an important role in granule cell development eventually regulating cerebellar network formation and motor control.

Interaction of p21(CDKN1A) with PCNA regulates the histone acetyltransferase activity of p300 in nucleotide excision repair.

The cell-cycle inhibitor p21(CDKN1A) has been suggested to directly participate in DNA repair, thanks to the interaction with PCNA. Yet, its role has remained unclear. Among proteins interacting with both p21 and PCNA, the histone acetyltransferase (HAT) p300 has been shown to participate in DNA repair. Here we report evidence indicating that p21 protein localizes and interacts with both p300 and PCNA at UV-induced DNA damage sites. The interaction between p300 and PCNA is regulated in vivo by p21. Indeed, loss of p21, or its inability to bind PCNA, results in a prolonged binding to chromatin and an increased association of p300 with PCNA, in UV-irradiated cells. Concomitantly, HAT activity of p300 is reduced after DNA damage. In vitro experiments show that inhibition of p300 HAT activity induced by PCNA is relieved by p21, which disrupts the association between recombinant p300 and PCNA. These results indicate that p21 is required during DNA repair to regulate p300 HAT activity by disrupting its interaction with PCNA.

Small ribosomal subunits associate with nuclear myosin and actin in transit to the nuclear pores.

We have followed at high resolution the ribosomal protein S6 entering the nucleus of HeLa cells, stopping in some (not all) interchromatin granules clusters and reaching, via Cajal bodies, the nucleolus. There, S6 is assembled with other proteins and rRNA into small ribosomal subunit (SSU), released in the nucleoplasm, and exported through the nuclear pores. We show for the first time the spatial association of nuclear myosin I (NMI) and actin with the SSU already at the nucleolar periphery to the nuclear pore. A blockade of NMI or actin induces an upstream accumulation of the S6 protein en route to the nucleolus, and a temperature lower than normal influences RNA export. Our data strongly suggest a functional relationship of SSU with NMI and actin. In our hypothesis, an active, myosin-driven movement of the small ribosomal subunit can be responsible for the export of approximately 10% of SSUs. This hypothesis is supported by ultrastructural, immunofluorescence, and biochemical analyses. The currently accepted model for the subunit release suggests a diffusive, temperature-independent mechanism. However, the advantage of the double mechanism would assure that the movement of a part of the subunits could be modulated, increased, or decreased according to the needs of the cell at a specific moment in the cell cycle.

Spatiotemporal dynamics of p21CDKN1A protein recruitment to DNA-damage sites and interaction with proliferating cell nuclear antigen.

The cyclin-dependent kinase inhibitor p21CDKN1A plays a fundamental role in the DNA-damage response by inducing cell-cycle arrest, and by inhibiting DNA replication through association with the proliferating cell nuclear antigen (PCNA). However, the role of such an interaction in DNA repair is poorly understood and controversial. Here, we provide evidence that a pool of p21 protein is rapidly recruited to UV-induced DNA-damage sites, where it colocalises with PCNA and PCNA-interacting proteins involved in nucleotide excision repair (NER), such as DNA polymerase delta, XPG and CAF-1. In vivo imaging and confocal fluorescence microscopy analysis of cells coexpressing p21 and PCNA fused to green or red fluorescent protein (p21-GFP, RFP-PCNA), showed a rapid relocation of both proteins at microirradiated nuclear spots, although dynamic measurements suggested that p21-GFP was recruited with slower kinetics. An exogenously expressed p21 mutant protein unable to bind PCNA neither colocalised, nor coimmunoprecipitated with PCNA after UV irradiation. In NER-deficient XP-A fibroblasts, p21 relocation was greatly delayed, concomitantly with that of PCNA. These results indicate that early recruitment of p21 protein to DNA-damage sites is a NER-related process dependent on interaction with PCNA, thus suggesting a direct involvement of p21 in DNA repair.

Both early and delayed treatment with melanocortin 4 receptor-stimulating melanocortins produces neuroprotection in cerebral ischemia.

Ischemic stroke is one of the main causes of death and disability. We investigated whether melanocortin peptides, which have protective effects in severe hypoxic conditions, also produce neuroprotection in a gerbil model of ischemic stroke. A 10-min period of global cerebral ischemia, induced by occluding both common carotid arteries, caused impairment in spatial learning and memory that was associated with activation of inflammatory and apoptotic pathways, including severe DNA damage and delayed neuronal death, in the hippocampus. Treatment with nanomolar doses of the melanocortin analog [Nle4, D-Phe7] alpha-MSH [which activates the melanocortin receptor subtypes (MC) mainly expressed in central nervous system, namely MC3 and MC4] modulated the inflammatory and apoptotic cascades and reduced hippocampus injuries even when delayed up to 9 h after ischemia, with consequent dose-dependent improvement in subsequent functional recovery. The selective MC3 receptor agonist gamma2-MSH had no protective effects. Pharmacological blockade of MC4 receptors prevented the neuroprotective effects of [Nle4, D-Phe7] alpha-MSH and worsened some ischemia outcomes. Together, our findings suggest that MC4 receptor-stimulating melanocortins might provide potential to develop a class of drugs with a broad treatment window for a novel approach to neuroprotection in ischemic stroke.

MPTP-induced increase in c-Fos- and c-Jun-like immunoreactivity in the monkey cerebellum.

The transcription factors c-Fos and c-Jun have been described to be overexpressed following many pathological stimuli, but whether they are required for neurodegeneration or neuroprotection is still open. In the present report, we analyzed the role of c-Fos and c-Jun proteins in Purkinje cell degeneration caused by the neurotoxin MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) in the monkey cerebellum, and determined the neuroprotective effect of the antioxidant drug a-dihydroergocryptine (DHEC), whose prior and simultaneous administration reduced the MPTP-induced neuronal loss in the substantia nigra. Immunocytochemistry for c-Fos- and c-Jun-like proteins showed persistent increased staining in Purkinje cells of MPTP-treated monkeys. The staining was greatly reduced in animals receiving DHEC. Similar results were observed in white matter glial cells after immunoreaction for c-Fos. The results suggest that, at least as far as the cerebellum is concerned, the increase in c-Fos and c-Jun expression correlate with cell damage, rather than with preservation.

Regional alterations of the NO/NOS system in the aging brain: a biochemical, histochemical and immunochemical study in the rat.

We have used several approaches (immunohistochemistry and enzyme histochemistry, Western blotting, biochemical assay of Ca(2+)-dependent catalytic activity) in order to detect differences in neuronal nitric oxide synthase (nNOS) expression and activity in various brain regions of young-adult (4-month-old) and aged (28-month-old) rats. In most of the brain regions examined (striatum, neocortex, olfactory cortex and hippocampus) some significant decrease in the density per unit area of nNOS neurons, detected either through immunohistochemistry or enzyme histochemistry, was observed in aged rats. However, only in the striatum and olfactory cortex this was accompanied by a significant decrease of the catalytic activity of the constitutive, Ca(2+)-dependent NOS form. In these two regions, the relative level of expression of nNOS protein was also significantly decreased, as assessed by Western blotting of proteic extracts from young-adult and aged rats. Other observed differences were a paler stain of neurons in some brain areas of the aged rats and differences of cellular compartmentalization of the protein in the same rats, as assessed through confocal microscopy. The present observations demonstrate that the expression and activity of nNOS show regionally-specific alterations in the brain of aged healthy rats, with a trend towards decrease, rather than toward increase as suggested by some previous reports. Therefore, hypotheses implicating nitric oxide increase in brain aging should be reconsidered on the basis of a clear-cut distinction between the physiological and the pathological aspects of the aging process.

The malformation of the cerebellar fissura prima: a tool for studying histogenetic processes.

Due to its regular cytoarchitecture and the relatively low number of cell types, the development of the cerebellar cortex is a model of election for studies of morphogenetic processes. To unravel the cellular and molecular mechanisms that regulate cell development, migration and differentiation and the settling of local circuits, pertubation of the three-layered organization of the cerebellar cortex has been induced by X-ray irradiation or antimitotic drug. In this review we deal with some data about the incidence and development of the malformation of the cerebellar fissura prima of the rat, as an eligible model for histogenetic studies. The naturally occurring malformation of the fissura prima is characterized by the loss of the three-layered organization of folia and the presence of large masses of ectopic granule cells. The malformation appears to be under genetic control, since the incidence of affected animals is consistent over extended breeding cycles, although the target of the eventual mutation is unknown. The observation of development of the malformation in infant rats suggests that a defect in meninges, and in particular in the pia mater, is a primary contributing factor. The molecules responsible for this defect are not identified, but they must be involved in basal lamina stabilization or destabilization. In fact, there is evidence that meninges do develop during first stages of histogenesis and only later degenerate. A possible correlation with certain human pathologies that involve defects in foliation is discussed.