RICERCA ITALIANA     

Research program

Role of metals – Ubiquitin/Proteasome interaction in the pathogenesis of conformational diseases

University Co-ordinator

Università degli Studi "G. d'Annunzio" CHIETI-PESCARA - ONCOLOGIA E NEUROSCIENZE - ()

Research Unit Leader

Stefano Sensi

Description

The present proposal will investigate the injurious role played by Zn2+ in the context of a pro-AD environment. To that aim, we intend to carry out studies where we apply advanced fluorescent imaging techniques and neurotoxicity essays to neurons and brain slices derived from wild type or AD-prone triple transgenic mice (3XTG-AD; Oddo et al., 2003). The 3XTG-AD mouse, over-expresseses h-tau (tauP301L), and mutated PS1 (PS1M146V) and APP (APPSwe), and virtually recapitulates the major pathological features of the AD brain. As reported, 3XTG-AD mice show signs of a progressive and age-dependent A-beta and tau pathology. Quite interestingly, AD-vulnerable CA1 pyramidal neurons of the TG mice undergo a very precocious (4-6 months of age) intracellular (but not extracellular) A-beta accumulation that well correlates with the concomitant appearance of signs of altered synaptic transmission and decreased Long-term Potentiation (Oddo et al., 2003;Billings et al., 2005). These exciting results strongly indicate a very prominent role for alterations in intracellular A-beta in the AD injurious cascade. The studies are centered around testing the following HYPOTHESIS: The injury promoting divalent cation, Zn2+ play crucial roles in AD related neuronal injury and is a critical link between the “pro-AD factors” and neurodegeneration in AD.

Understanding the reciprocal interactions between the factors and this ion will allow us to test new therapeutic approaches focused on restoring Zn2+ homeostasis and counteracting the neurotoxic actions of this cation.

Questions to be answered are as follows:

SPECIFIC AIM I: Zn2+ homeostasis in 3XTG-AD neurons
A. Is Zn2+ homeostasis deranged in 3XTG-AD neurons?
High resolution imaging (fig.1) will be employed to investigate Zn2+ dynamics (particularly, in subcellular compartments including the ER and mitochondria), in cultured 3XTG-AD and control neurons. Effects of Zn2+ influx and intracellular release will be studied. [Zn2+]i rises will be here evoked by different patho-physiological triggers (high K+-triggered depolarization, sublethal glutamate receptor activation, MTs oxidation, mitochondrial depolarization)
B. Is Zn2+ homeostasis perturbed in aging 3XTG-AD neurons?
A parallel set of studies will examine alterations in Zn2+ homeostasis in aged (6-8 weeks old cultures) 3XTG-AD neurons.
C. Is MT expression altered in 3XTG-AD mice
Using young, adult and aged 3XTG-AD mice we will test the expression of MTs (MT-1/2 and MT-3) as well as the topographical distribution of Zn2+ to ascertain the possible link between Zn2+ dismetabolism and MTs expression.

SPECIFIC AIM II: How do cell culture studies translate to the “in vivo” scenario?
Zn2+ homeostasis in 3XTG- AD neurons from brain slice preparations. While cell culture models are useful for studying mechanisms, under controlled and reproducible conditions, experiments in tissues preserving native connectivity and architecture are necessary to verify the degree to which these processes are likely to impact injury in vivo. To that aim, we will also explore [Zn2+]i homeostasis in neurons obtained from acute brain slice preparations from both young and aging 3XTG-AD and non-TG mice.

A. Zn2+ imaging of hippocampal pyramidal neurons (HPN)s in brain slices from 3XTG-AD and control mice: Confocal Zn2+ imaging of CA1 HPNs will be performed on acute coronal brain slices (200-400um) obtained from 4-6 weeks old TG and non-TG mice loaded with the ester form of high and low affinity Zn2+ probes such as Fluozin-3 or Newport Green. Initial studies, using the cell permeable form of the selected Zn2+ indicator, will visualize neurons after dye loading of the entire slice. As this approach might result in poor spatial discrimination of the fluorescence signal, in further trials we will try to dye load single pyramidal neurons by pulse injection of the acid form of the specific probes (Gorter et al., 1997). Once dye is loaded, slices will be placed on the stage of a confocal microscope and kept under constant perfusion of a physiological buffer bubbled with 95%O2;5%CO2. Slices will then be challenged with Zn2+ loads triggered by the same maneuvers described above (Specific Aim I.A).
B. Zn2+ imaging in aging mature tissue: We will address the issue of Zn2+ dyshomeostasis in aging (and AD-prone) neurons by repeating the same set of experiments described above in acute brain slice preparations obtained from “aging” (12-24 months old) 3XTG-AD and control animals.

SPECIFIC AIM III: What are the injurious downstream consequences of Zn2+ dyshomeostasis?
A. Zn2+ dyshomeostasis and A-beta. Does [Zn2+]i accumulation promote intracellular A-beta aggregation?
As discussed in the background section, Zn2+ critically modulates A-beta aggregation and plaque formation (Bush et al., 1994; Huang et al., 1997; Bush, 2003; Danscher et al., 1997; Suh et al., 2000). It is conceivable that [Zn2+]i accumulation inside neurons can facilitate the early formation of an intracellular nidus of A-beta aggregation. To test this hypothesis, we will therefore try to correlate [Zn2+]i rises with the early appearance of A-beta aggregates in the cytosol of 3XTG-AD neurons from both cultures and brain slices.
In this section, the appearance of aggregates will be evaluated using immunoprecipitation, western blotting and staining with conformation specific antibodies. These antibodies, obtained through collaboration with Dr. Charlie Glabe, are specific for amyloid oligomers or “prefibrillar aggregates” and should work quite effectively to investigate an early stage appearance of such A-beta oligomers. This line of experiments will be performed on both cultured 3XTG-AD neurons and 3XTG-AD CA1 HPNs from acute brain slices. We will start by studying effects of exogenous Zn2+ loading in neurons exposed to Zn2+ influx (5-10 min exposure to high-K+ with Zn2+ 100-300 uM), and the presence of aggregates will be evaluated at 10, 30, 60 and 180 minutes after these Zn2+ loads. We will also explore whether intracellular Zn2+ mobilization from intracellular stores can promote A-beta aggregation in neurons exposed to DTDP and FCCP (to promote intracellular Zn2+ release from MTs and mitochondria, respectively). Finally, we will examine how manipulation of Zn2+ homeostatic mechanisms might alter the aggregation process. To that aim, we will test how intracellular Zn2+ chelation (TPEN, clioquinol, DP-109, carnosine and our own carnosine derivatives) can impact the process. We will also evaluate whether inhibition of Zn2+ release from endogenous stores by using either antioxidants [vitamin E derivatives, the SOD mimetic antioxidant, Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP; Patel, et al., 1996)] to block redox-sensitive Zn2+ release from MTs), or mPTP blockers (cyclosporine A, bongkrekic acid, to prevent mitochondrial Zn2+ efflux), can affect intracellular A-beta aggregation.

B. Evolution of synaptic derangement and effects of protective interventions
Findings on 3XTG-AD mice indicate that synaptic dysfunction may be a critical contributing factor in AD (Selkoe 2002; Oddo et al., 2003; Billings et al., 2005). In AD, areas of A-beta deposition are associated with dystrophic neurites and these dystrophic processes correlate well with the severity of cognitive impairment (McKee et al., 1991). We will employ an “in vivo” multiphoton imaging system (see fig.2) to monitor A-beta–associated dystrophic neurites in the brains of living 3XTG-AD crossed with Thy-1-YFP transgenic mice (Thy-1-YFP mice express YFP in the neuronal somata and dendrites of some cortical neurons; Feng et al.,2000; Brendza et al., 2005). In this section we will also investigate whether different intervention strategies could rescue neuritic dystrophy and reduce A-beta plaque load in 3XTG-AD/Thy-1-YFP mice.

i: Time course of the neuritic derangement in 3XTG-AD/Thy-1-YFP mice: 3XTG-AD/Thy-1-YFP mice will be imaged at different ages (2,4,6,12 months) to investigate the distribution, number and volume of dendritic varicosities associated with A-beta plaques marked with methoxy-X04.

ii. Exploring neuroprotective strategies: Zn2+ chelation: Previous studies in APP Tg2576 transgenic mice show that protracted treatment with Clioquinol (CQ; an orally bioavailable Cu–Zn chelator) or DP-109 (a lipophilic Zn2+ chelator) promote a dramatic decrease in amyloid plaque load (Cherny et al., 2001; Lee et al., 2004). Carnosine is a cell-permeable Zn2+ and Cu2+ chelator that offers the advantage of being an excellent antioxidant as well. Here we will test whether chronic treatment with DP109, Clioquinol or carnosine (and our own carnosine derivatives) reduces A-beta plaque load and promotes the recovery of neuritic dystrophy in 3XTG-AD/Thy-1-YFP mice. The chelators will be given chronically per os (DP-109 or CQ; Nitzan et al., 2003; Lee et al., 2004) or IP (carnosine) and changes in neurite morphology and A-beta plaque deposits examined using 2 photon imaging in animals treated at different age (2, 4, 6, 12 months).

iii. Exploring neuroprotective strategies – modulation of proteasome activity: Compounds synthesized by the Catania and Naples Units will be tested for their efficacy in reverting the proteasome inhibition induced by A-beta, h-tau, and Zn2+. In these experiments we will test whether chronic treatment with these compounds could promote increased activity of the proteasome complex and be neuroprotective in an animal model of AD. We will start using neuronal cell cultures testing whether the pro-AD environment of 3XTG-AD mice leads to decrease proteasome activity. In order to detect the change in the proteasome activity neurons will be transfected with GFP constructs (pEGFP-C1 or pEGFP-C1-CL1). CL1, is a degradation signal and confers instability to pEGFP leading to rapid (30 min) entry of the construct into the proteasome pathway (Gilon et al., 1998; Kain, 1999) while pEGFP-C1 is highly stable, resistant to degradation and used as control. We will test fluorescence changes of pEGFP-C1-CL1 overtime, where inhibition of proteasome activity is expected to result in increased fluorescence of the construct. After evaluating baseline inhibition of proteasome activity in 3XTG-AD neurons we will move to test whether our experimental compounds could revert the process.
In the second stage of the project, 3XTG-AD/Thy-1-YFP animals will be treated with the compounds tested in neuronal cultures and chymotrypsin-like activity (an index of ubiquitin–proteasome activity; Heinemeyer et al., 1991), measured. Along with measurements of proteasome activity, signs of neuritic dystrophy and Ab plaque load will be assessed as above at different ages.

Summary and Significance
We aim to achieve a better understanding of the mechanisms involved in the AD related neuronal loss. We anticipate that the proposed experiments, performed in a setting (aging, 3XTG-AD mice) that more closely mimics the degenerative processes occurring in vivo, will further our knowledge of the injurious “feed forward” cycle, described above, and eventually result in new therapeutic approaches. Questions to be specifically addressed include: How does a pro-AD environment affect [Zn2+]i homeostasis? Which are the subcellular sites involved in Zn2+ deregulation? Is Zn2+ dyshomeostasis promoting an early stage of A-beta aggregation intracellularly? Can Zn2+ dyshomeostasis set in motion early stage synapse disruption? Can Zn2+ chelation (or modulation of Zn2+ inhibition on the proteasome activity) reverse some of the early pathogenic changes?

General Procedures:
Neuronal Culture: Mixed dissociated cortical or hippocampal cell cultures are prepared from fetal 3XTG-AD and control mice (15-16 days), and plated on a previously established bed of cortical or hippocampal glia, as previously described (Yin et al., 1998). Cultures plating density varied between about 0.2-2 x 105 cells/cm. For imaging studies, cells are plated at the lower densities on poly-lysine and laminin coated glass coverslips. Cultures are studied after 13-28 days in vitro.
Assessment of cell survival: Neuronal cell injury is assessed in culture using direct examination supplemented either by biochemical assays (eg. meaurement of lactate dehydrogenase (LDH), released into the media by damaged cells), nuclear morphology (eg bisbenzamide staining), or “cell death” stains [trypan blue, propidium iodide (PI)].
Immunohistochemistry: To analyze A-beta aggregation, paraformaldehyde-fixed, 50 um brain sections, or cultured cortical and hippocampal neurons are immunostained using anti-A-beta 42 antibodies (provided by Dr.Glabe). Primary antibodies are applied at dilutions of 1:200 and cultures or slices developed with diaminobenzidine (DAB) substrate using the avidin-biotin horseradish peroxidase system.
Fluorescent imaging: Dye loaded or GFP trasnfected neuronal cultures on glass bottom dishes are put on the stage of a Nikon Diaphot inverted microscope (with 75 W Xenon Lamp, Nikon 40X or 100X, 1.3 N.A. epifluorescence oil immersion objectives. Illumination is with a computer controlled filter wheel fitted with excitation filters appropriate to the dye, emission recorded (after passage through a dichroic mirror and emission filter) by a 12 bit cooled CCD camera, and analyzed using Metafluor software . Background fluorescence from a cell free region is subtracted. Depending on dye type and experimental purpose, values are presented as fluorescence ratios or calibrated values, or are normalized to basal fluorescence values for the same cell (fig.1 ). Confocal imaging is performed on the stage of a 12 bit scanning confocal microscope (Zeiss Meta)equipped with Argon, Helium/Neon I, and Helium/Neon II lasers (excitations at 457,488, 514, 543 o 633 nm, emissions between 500 e 700 nm). To avoid phototoxicity, the laser is attenuated (6% of maximum). Images are analyzed with Zeiss custom software and ImageJ (NIH). For slice studies, imaging is carried out at 30-35°C under continuous perfusion with oxygenated (95% 02 /5% C02) buffer.
Slice preparation (Yin et al., 2002): Upon vibratome sectioning (200-400 um), coronal slices are transferred to chamber containing cold oxygenated equilibration buffer (consisting of, in mM, 126 NaCl, 24 NaHCO3, 1 NaH2PO4, 2.5 KCl, 10 MgSO4, 10 glucose, pH 7.4), containing MK-801, Gd3+ (20 uM) and NAS; 300 uM. After 25 min, slices are washed before transfer to the microscope chamber for confocal or conventional imaging.
Multiphoton imaging in living animals: 3XTG-AD/Thy-1-YFP mice, are anesthetized and a portion of the skull and the dura removed from the exposed brain region. A permanent optical window is obtained by covering the exposed brain region with an 8-mm cover glass sealed to the surrounding skull with dental cement (fig.2 ). Experiments are done in collaboration with the multiphoton facility at LENS. During the imaging session, anesthetized animals are placed on a stereotaxic device (Stoelting Co.) mounted on the stage of our 2-photon custom made microscope with a Chameleon Ti:Sa laser (Coherent Inc.) Excitation wavelength is 935 nm and emission is collected at 550 nm. Low-magnification images (obtained using an Olympus XLUMPlanFl 20x 0.95 W objective) are taken to establish the positions of the neurons and individual dendrite spines imaged. Images are acquired as 512 × 512 pixel of 12-bit pixels in z-series stacks originating at the cortical surface.
Assessment of MT espression:
MT expression will be determined immunohistochemically as previously reported (Zatta et al.,2006). The quantification of MTs in tissues and cell cultures will be carried out by using the silver saturation method reported by Scheuhammer and Cherian, (1991).
Assessment of brain Zn2+:
Brain Zn2+ will be determined by atomic absorption spectrophotometry (Zatta et al.,2005 and 2006).