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Task-free functional connectivity in animal models provides an experimental framework to examine connectivity phenomena under controlled conditions and allows for comparisons with data modalities collected under invasive or terminal procedures. Currently, animal acquisitions are performed with varying protocols and analyses that hamper result comparison and integration. Here we introduce StandardRat, a consensus rat functional magnetic resonance imaging acquisition protocol tested across 20 centers. To develop this protocol with optimized acquisition and processing parameters, we initially aggregated 65 functional imaging datasets acquired from rats across 46 centers. We developed a reproducible pipeline for analyzing rat data acquired with diverse protocols and determined experimental and processing parameters associated with the robust detection of functional connectivity across centers. We show that the standardized protocol enhances biologically plausible functional connectivity patterns relative to previous acquisitions. The protocol and processing pipeline described here is openly shared with the neuroimaging community to promote interoperability and cooperation toward tackling the most important challenges in neuroscience., (© 2023. The Author(s), under exclusive licence to Springer Nature America, Inc.)

The multiplicity of systems affected in Alzheimer's disease (AD) brains calls for multi-target therapies. Although mesenchymal stem cells (MSC) are promising candidates, their clinical application is limited because of risks related to their direct implantation in the host. This could be overcome by exploiting their paracrine action. We herein demonstrate that in vivo systemic administration of secretome collected from MSC exposed in vitro to AD mouse brain homogenates (MSC-CS), fully replicates the cell-mediated neuroreparative effects in APP/PS1 AD mice. We found a complete but transient memory recovery by 7 days, which vanished by 14 days, after a single MSC-CS intravenous administration in 12-month or 22-24-month-old mice. Treatment significantly reduced plaque load, microglia activation, and expression of cytokines in astrocytes in younger, but not aged, mice at 7 days. To optimize efficacy, we established a sustained treatment protocol in aged mice through intranasal route. Once-weekly intranasal administration of MSC-CS induced persistent memory recovery, with dramatic reduction of plaques surrounded by a lower density of β-amyloid oligomers. Gliosis and the phagocytic marker CD68 were decreased. We found a higher neuronal density in cortex and hippocampus, associated with a reduction in hippocampal shrinkage and a longer lifespan indicating healthier conditions of MSC-CS-treated compared to vehicle-treated APP/PS1 mice. Our data prove that MSC-CS displays a great multi-level therapeutic potential, and lay the foundation for identifying the therapeutic secretome bioreactors leading to the development of an efficacious multi-reparative cocktail drug, towards abrogating the need for MSC implantation and risks related to their direct use.

Alzheimer's disease (AD), the most common form of dementia, is a progressive neurodegenerative disorder associated with aberrant production of beta-amyloid (Aβ) peptide depositing in brain as amyloid plaques. While animal models allow investigation of disease progression and therapeutic efficacy, technology to fully dissect the pathological mechanisms of this complex disease at cellular and vascular levels is lacking. X-ray phase contrast tomography (XPCT) is an advanced non-destructive 3D multi-scale direct imaging from the cell through to the whole brain, with exceptional spatial and contrast resolution. We exploit XPCT to simultaneously analyse disease-relevant vascular and neuronal networks in AD mouse brain, without sectioning and staining. The findings clearly show the different typologies and internal structures of Aβ plaques, together with their interaction with patho/physiological cellular and neuro-vascular microenvironment. XPCT enables for the first time a detailed visualization of amyloid-angiopathy at capillary level, which is impossible to achieve with other approaches. XPCT emerges as added-value technology to explore AD mouse brain as a whole, preserving tissue chemistry and structure, enabling the comparison of physiological vs. pathological states at the level of crucial disease targets. In-vivo translation will permit to monitor emerging therapeutic approaches and possibly shed new light on pathological mechanisms of neurodegenerative diseases., (Copyright © 2018. Published by Elsevier Inc.)

Auditory "oddball" event-related potentials (aoERPs), resting state functional magnetic resonance imaging (rsfMRI) connectivity, and electroencephalographic (rsEEG) rhythms were tested as longitudinal functional biomarkers of prodromal Alzheimer's disease (AD). Data were collected at baseline and four follow-ups at 6, 12, 18, and 24 months in amnesic mild cognitive impairment (aMCI) patients classified in two groups: "positive" (i.e., "prodromal AD"; n = 81) or "negative" (n = 63) based on a diagnostic marker of AD derived from cerebrospinal samples (Aβ42/P-tau ratio). A linear mixed model design was used to test functional biomarkers for Group, Time, and Group×Time effects adjusted by nuisance covariates (only data until conversion to dementia was used). Functional biomarkers that showed significant Group effects ("positive" versus "negative", p < 0.05) regardless of Time were 1) reduced rsfMRI connectivity in both the default mode network (DMN) and the posterior cingulate cortex (PCC), both also giving significant Time effects (connectivity decay regardless of Group); 2) increased rsEEG source activity at delta (<4 Hz) and theta (4-8 Hz) rhythms and decreased source activity at low-frequency alpha (8-10.5 Hz) rhythms; and 3) reduced parietal and posterior cingulate source activities of aoERPs. Time×Group effects showed differential functional biomarker progression between groups: 1) increased rsfMRI connectivity in the left parietal cortex of the DMN nodes, consistent with compensatory effects and 2) increased limbic source activity at theta rhythms. These findings represent the first longitudinal characterization of functional biomarkers of prodromal AD relative to "negative" aMCI patients based on 5 serial recording sessions over 2 years.

Repurposing doxycycline for the treatment of amyloidosis has recently been put forward because of the antiaggregating and anti-inflammatory properties of the drug. Most of the investigations of the therapeutic potential of doxycycline for neurodegenerative amyloidosis, e.g., prion and Alzheimer disease (AD), have been carried out in mouse models, but surprisingly no data are available regarding the concentrations reached in the brain after systemic administration. We filled this gap by analyzing the pharmacokinetic profile of doxycycline in plasma and brain after single and repeated intraperitoneal injections of 10 and 100 mg/kg, in wild-type mice and the APP23 mouse model of AD. The main outcomes of our study are: 1) Peak plasma concentrations ranged from 2 to10 μ g/ml, superimposable to those in humans; 2) brain-to-plasma ratio was ∼0.2, comparable to the cerebrospinal fluid/serum ratios in humans; 3) brain C max 4-6 hours after a single dose was ∼0.5 (10 mg/kg) and ∼5 μ M (100 mg/kg). Notably, these concentrations are lower than those required for the drug's antiaggregating properties as observed in cell-free studies, suggesting that other features underlie the positive cognitive effects in AD mice; 4) elimination half-life was shorter than in humans (3-6 vs. 15-30 hours), therefore no significant accumulation was observed in mouse brain following repeated treatments; and 5) there were no differences between doxycycline concentrations in brain areas of age-matched wild-type and APP23 mice. These data are useful for planning preclinical studies with translational validity, and to identify more reliably the mechanism(s) of action underlying the central in vivo effects of doxycycline., (Copyright © 2018 by The American Society for Pharmacology and Experimental Therapeutics.)

Background: Early Alzheimer's disease (AD) detection using cerebrospinal fluid (CSF) biomarkers has been recommended as enrichment strategy for trials involving mild cognitive impairment (MCI) patients., Objective: To model a prodromal AD trial for identifying MRI structural biomarkers to improve subject selection and to be used as surrogate outcomes of disease progression., Methods: APOE ɛ4 specific CSF Aβ42/P-tau cut-offs were used to identify MCI with prodromal AD (Aβ42/P-tau positive) in the WP5-PharmaCog (E-ADNI) cohort. Linear mixed models were performed 1) with baseline structural biomarker, time, and biomarker×time interaction as factors to predict longitudinal changes in ADAS-cog13, 2) with Aβ42/P-tau status, time, and Aβ42/P-tau status×time interaction as factors to explain the longitudinal changes in MRI measures, and 3) to compute sample size estimation for a trial implemented with the selected biomarkers., Results: Only baseline lateral ventricle volume was able to identify a subgroup of prodromal AD patients who declined faster (interaction, p = 0.003). Lateral ventricle volume and medial temporal lobe measures were the biomarkers most sensitive to disease progression (interaction, p≤0.042). Enrichment through ventricular volume reduced the sample size that a clinical trial would require from 13 to 76%, depending on structural outcome variable. The biomarker needing the lowest sample size was the hippocampal subfield GC-ML-DG (granule cells of molecular layer of the dentate gyrus) (n = 82 per arm to demonstrate a 20% atrophy reduction)., Conclusion: MRI structural biomarkers can enrich prodromal AD with fast progressors and significantly decrease group size in clinical trials of disease modifying drugs.

β-Amyloid oligomers (AβOs) and neuroinflammation are 2 main culprits to counteract in Alzheimer's disease (AD). Doxycycline (DOXY) is a second generation antibiotic of the tetracycline class that are promising drugs tested in many clinical trials for a number of different pathologies. DOXY is endowed with antiamyloidogenic properties and better crosses the blood-brain barrier, but its efficacy has never been tested in AD mice. We herein show that 15- to 16-month-old APP/PS1dE9 (APP/PS1) AD mice receiving DOXY under different treatment regimens recovered their memory without plaque reduction. An acute DOXY treatment was, also, sufficient to improve APP/PS1 mouse memory, suggesting an action against soluble AβOs. This was confirmed in an AβO-induced mouse model, where the AβO-mediated memory impairment was abolished by a DOXY pretreatment. Although AβOs induce memory impairment through glial activation, assessing the anti-inflammatory action of DOXY, we found that in both the AβO-treated and APP/PS1 mice, the memory recovery was associated with a lower neuroinflammation. Our data promote DOXY as a hopeful repositioned drug counteracting crucial neuropathological AD targets., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Glucose is the central nervous system's only energy source. Imaging techniques capable to detect pathological alterations of the brain metabolism are useful in different diagnostic processes. Such techniques are also beneficial for assessing the evaluation efficacy of therapies in pre-clinical and clinical stages of diseases. Chemical exchange saturation transfer (CEST) magnetic resonance imaging (MRI) is a possible alternative to positron emission tomography (PET) imaging that has been widely explored in cancer research in humans and animal models. We propose that pathological alterations in brain 2-deoxy-D-glucose (2DG) uptake, typical of neurodegenerative diseases, can be detected with CEST MRI. Transgenic mice overexpressing a mutated form of amyloid precusrsor protein (APP23), a model of Alzheimer's disease, analyzed with CEST MRI showed a clear reduction of 2DG uptake in different brain regions. This was reminiscent of the cerebral condition observed in Alzheimer's patients. The results indicate the feasibility of CEST for analyzing the brain metabolic state, with better image resolution than PET in experimental models.

The failure of clinical trials largely focused on mild to moderate stages of Alzheimer disease has suggested to the scientific community that the effectiveness of Amyloid-β (Aβ)-centered treatments should be evaluated starting as early as possible, well before irreversible brain damage has occurred. Accordingly, also the preclinical development of new therapies should be carried out taking into account this suggestion. In the present investigation we evaluated the efficacy of a treatment with liposomes multifunctionalized for crossing the blood-brain barrier and targeting Aβ, carried out on young APP/PS1 Tg mice, taken as a model of pre-symptomatic disease stage. Liposomes were administered once a week to Tg mice for 7months, starting at the age of 5months and up to the age of 12 when they display AD-like cognitive and brain biochemical/anatomical features. The treatment prevented the onset of the long-term memory impairment and slowed down the deposition of brain Aβ; at anatomical level, prevented both ventricle enlargement and entorhinal cortex thickness reduction, otherwise occurring in untreated mice. Strikingly, these effects were maintained 3months after treatment discontinuation. An increase of Aβ levels in the liver was detected at the end of the treatment, then followed also by reduction of brain Amyloid Precursor Protein and increase of Aβ-degrading enzymes. These results suggest that the treatment promotes brain Aβ clearance by a peripheral 'sink' effect and ultimately affects Aβ turnover in the brain. Worth of note, the treatment was apparently not toxic for all the organs analyzed, in particular for brain, as suggested by the lower brain TNF-α and MDA levels, and by higher level of SOD activity in treated mice. Together, these findings promote a very early treatment with multi-functional liposomes as a well-tolerated nanomedicine-based approach, potentially suitable for a disease-modifying therapy of AD, able to delay or prevent relevant features of the disease., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Alzheimer's disease (AD) is the most common form of dementia characterized by synaptic dysfunction, memory loss, neuroinflammation, and neuronal cell death. Amyloid-β (Aβ), recognized as the main culprit of AD, aggregates and accumulates in the extracellular compartment as neuritic plaques, after deregulation of its production or clearance. Apolipoprotein E (ApoE) plays a major role in Aβ clearance and its expression is transcriptionally regulated by the liver X receptor and retinoid X receptors (RXRs) system. Bexarotene (BEXA), an RXR agonist that increases ApoE expression and microglia phagocytosis has been proposed as a promising therapy for AD, resolving both the amyloid pathology and memory loss. Despite the first compelling report, however, multiple failures have been documented, raising concern about whether BEXA could in fact become a novel disease-modifying strategy for AD. To help clarify this, we investigated the effect of BEXA in vivo at multiple levels in TASTPM transgenic mice. Seven-day oral administration of BEXA to these mice did not achieve any significant memory improvement, plaque reduction, or enhancement of microglial cell activation. No differences were found when specifically investigating the microglial phagocytic state in vivo. In addition, a brain structural analysis with magnetic resonance did not detect any BEXA-mediated change in the volume reduction of the main affected brain areas in our mice. These results suggest that BEXA has no beneficial effects on the multi-factorial pathologic phenotype of AD mice.

Environmental enrichment (EE) is a non-pharmacological intervention reported to counteract pathological signs in models of Alzheimer's disease (AD). We developed EE protocols in APP23 mice and evaluated how they influenced cognitive decline and brain amyloid-β (Aβ) burden. We also investigated the involvement of sirtuins (SIRTs) as a possible molecular mediator of EE, by assessing hippocampal and cortical mRNA and protein levels of the SIRT family members (SIRT1 to SIRT7). APP23 transgenic mice were moved to EE cages (TG-EEs) starting from 3 months of age. TG-EEs were compared to transgenic mice housed in standard cages (TG-SHs) and to wild-type littermates in the two housing conditions (WT-EEs and WT-SHs). At 7 months of age, all mice were tested for behavioral performance with Morris Water Maze (MWM) and visual novel Object Recognition Test (vORT). After a month, a group underwent biochemical analyses, while another group continued in the EE environment till 18 months of age, when Aβ plaque load was assessed. At 7 months, TG-SHs had impaired behavioral performance in MWM and vORT. In contrast, TG-EE mice had restored behavioral performance. At 8 months, EE did not affect AβPP expression or processing, Aβ40/42, pGlu-Aβ3-40/3-42, or Aβ oligomer level. The expression of two Aβ degrading enzymes (insulin degrading enzyme and neprilysin) was not modulated by EE. Brain sirtuin mRNA and protein levels were unchanged, while brain-derived neurotrophic factor expression increased after EE. Aβ deposition was attenuated in 18-month-old TG-EE mice, without apparent reduction of neuroinflammatory signs. We suggest that EE had a beneficial effect on cognitive performance and lessened long-term Aβ accumulation, but brain sirtuin expression was not modulated when cognitive impairment was restored.

Background: Developed countries are experiencing an unprecedented increase in life expectancy that is accompanied by a tremendous rise in the number of people with dementia. The purpose of this paper is to report on the study design and methodology of an Italian population-based study on brain aging and dementia in the elderly. This multi-domain study is structured in two phases. Our goal is to gather sufficient data to estimate the prevalence (phase I: cross-sectional study), the incidence and the progression of dementia and its subtypes as well as cognitive impairment (phase II: follow-up study) and to identify socio-demographic, clinical, and lifestyle factors associated with dementia and the quality of brain aging in people aged 70-74 years, a crucial point between late adulthood and old age., Methods/design: We chose to contact all 1773 people born between 1935-39 residing in Abbiategrasso, Milan, Italy. Those who agreed to participate in the "Invece.Ab" study were enrolled in a cross-sectional assessment and will be contacted two and four years after the initial data collection to participate in the longitudinal survey. Both the cross-sectional and longitudinal assessments include a medical evaluation, a neuropsychological test battery, several anthropometric measurements, a social and lifestyle interview, blood analyses, and the storage of a blood sample for the evaluation of putative biological markers., Discussion: Now at the end of the recruitment phase, the evaluable population has amounted to 1644 people. Among these, 1321 (80.35%) of the participants have completed phase I. This high return rate was likely due to the style of recruitment and personalization of the contacts.

Unlabelled: Follow-up of β-amyloid (Aβ) deposition in transgenic mouse models of Alzheimer disease (AD) would be a valuable translational tool in the preclinical evaluation of potential antiamyloid therapies. This study aimed to evaluate the ability of the clinically used PET tracer (11)C-Pittsburgh compound B ((11)C-PIB) to detect changes over time in Aβ deposition in the brains of living mice representing the APP23, Tg2576, and APP(swe)-PS1(dE9) transgenic mouse models of AD., Methods: Mice from each transgenic strain were imaged with 60-min dynamic PET scans at 7-9, 12, 15, and 18-22 mo of age. Regional (11)C-PIB retention was quantitated as distribution volume ratios using Logan graphical analysis with cerebellar reference input, as radioactivity uptake ratios between the frontal cortex (FC) and the cerebellum (CB) during the 60-min scan, and as bound-to-free ratios in the late washout phase (40-60 min). Ex vivo autoradiography experiments were performed after the final imaging session to validate (11)C-PIB binding to Aβ deposits. Additionally, the presence of Aβ deposits was evaluated in vitro using staining with thioflavin-S and Aβ1-40, Aβ1-16, and AβN3(pE) immunohistochemistry., Results: Neocortical (11)C-PIB retention was markedly increased in old APP23 mice with large thioflavin-S-positive Aβ deposits. At 12 mo, the Logan distribution volume ratio for the FC was 1.03 and 0.93 (n = 2), increasing to 1.38 ± 0.03 (n = 3) and 1.34 (n = 1) at 18 and 21 mo of age, respectively. An increase was also observed in bound-to-free ratios for the FC between young (7- to 12-mo-old) and old (15- to 22-mo-old) APP23 mice. Binding of (11)C-PIB to Aβ-rich cortical regions was also evident in ex vivo autoradiograms of APP23 brain sections. In contrast, no increases in (11)C-PIB retention were observed in aging Tg2576 or APP(swe)-PS1(dE9) mice in vivo, although in the latter, extensive Aβ deposition was already observed at 9 mo of age with immunohistochemistry., Conclusion: The results suggest that (11)C-PIB binding to Aβ deposits in transgenic mouse brain is highly dependent on the AD model and the structure of its Aβ plaques. Longitudinal in vivo (11)C-PIB uptake studies are possible in APP23 mice.

Different kinds of challenge can alter spontaneous ongoing electroencephalographic (EEG) rhythms in animal models, thus providing paradigms to evaluate treatment effects in drug discovery. The effects of challenges represented by pharmacological agents, hypoxia, sleep deprivation and transcranial magnetic stimulation (TMS) on EEG rhythms are here reviewed to build a knowledge platform for innovative translational models for drug discovery in Alzheimer's disease (AD). It has been reported that antagonists of cholinergic neurotransmission cause synchronisation of spontaneous ongoing EEG rhythms in terms of enhanced power of EEG low frequencies and decreased power of EEG high frequencies. Acetylcholinesterase inhibitors and serotonergic drugs may restore a normal pattern of EEG desynchronisation. Sleep deprivation and hypoxia challenges have also been reported to elicit abnormal synchronisation of spontaneous ongoing EEG rhythms in rodents. The feasibility and reproducibility of TMS have been demonstrated in rodents but information on a consistent modulation of EEG after TMS manipulation is very limited. Transgenic mice over-expressing human amyloid precursor protein complementary DNAs (cDNAs) harbouring the 'Swedish' mutation and PS-1 cDNAs harbouring the A264E mutation, which recapitulate some of the pathological features of AD, exhibit alterations of spontaneous ongoing EEG rhythms at several low and high frequencies. This does not appear, however, to be a consequence of beta-amyloid deposition in the brain. The present review provides a critical evaluation of changes of spontaneous ongoing EEG rhythms due to the experimental manipulations described above, in order to stimulate the promote more adherent models fitting dynamics in humans., (Copyright © 2012 International Federation of Clinical Neurophysiology. Published by Elsevier Ireland Ltd. All rights reserved.)

The concept of "prion-like" has been proposed to explain the pathogenic mechanism of the principal neurodegenerative disorders associated with protein misfolding, including Alzheimer disease (AD). Other evidence relates prion protein with AD: the cellular prion protein (PrP(C)) binds β amyloid oligomers, allegedly responsible for the neurodegeneration in AD, mediating their toxic effects. We and others have confirmed the high-affinity binding between β amyloid oligomers and PrP(C), but we were not able to assess the functional consequences of this interaction using behavioral investigations and in vitro tests. This discrepancy rather than being resolved with the classic explanations, differencies in methodological aspects, has been reinforced by new data from different sources. Here we present data obtained with PrP antibody that not interfere with the neurotoxic activity of β amyloid oligomers. Since the potential role of the PrP(C) in the neuronal dysfunction induced by β amyloid oligomers is an important issue, find reasonable explanation of the inconsistent results is needed. Even more important however is the relevance of this interaction in the context of the disease, so as to develop valid therapeutic strategies.

Stem Cell (SC) therapy is one of the most promising approaches for the treatment of Amyotrophic Lateral Sclerosis (ALS). Here we employed Super Paramagnetic Iron Oxide nanoparticles (SPIOn) and Hoechst 33258 to track human Amniotic Fluid Cells (hAFCs) after transplantation in the lateral ventricles of wobbler (a murine model of ALS) and healthy mice. By in vitro, in vivo and ex vivo approaches we found that: 1) the main physical parameters of SPIOn were maintained over time; 2) hAFCs efficiently internalized SPIOn into the cytoplasm while Hoechst 33258 labeled nuclei; 3) SPIOn internalization did not alter survival, cell cycle, proliferation, metabolism and phenotype of hAFCs; 4) after transplantation hAFCs rapidly spread to the whole ventricular system, but did not migrate into the brain parenchyma; 5) hAFCs survived for a long time in the ventricles of both wobbler and healthy mice; 6) the transplantation of double-labeled hAFCs did not influence mice survival.

Nephrin is an immunoglobulin-like adhesion molecule first discovered as a major component of the podocyte slit diaphragm, where its integrity is essential to the function of the glomerular filtration barrier. Outside the kidney, nephrin has been shown in other restricted locations, most notably in the central nervous system (CNS) of embryonic and newborn rodents. With the aim of better characterizing nephrin expression and its role in the CNS of adult rodents, we studied its expression pattern and possible binding partners in CNS tissues and cultured neuronal cells and compared these data to those obtained in control renal tissues and podocyte cell cultures. Our results show that, besides a number of locations already found in embryos and newborns, endogenous nephrin in adult rodent CNS extends to the pons and corpus callosum and is expressed by granule cells and Purkinje cells of the cerebellum, with a characteristic alternating expression pattern. In primary neuronal cells we find nephrin expression close to synaptic proteins and demonstrate that nephrin co-immunoprecipitates with Fyn kinase, glutamate receptors and the scaffolding molecule PSD95, an assembly that is reminiscent of those made by synaptic adhesion molecules. This role seems to be confirmed by our findings of impaired maturation and reduced glutamate exocytosis occurring in Neuro2A cells upon nephrin silencing. Of note, we disclose that the very same nephrin interactions occur in renal glomeruli and cultured podocytes, supporting our hypothesis that podocytes organize and use similar molecular intercellular signalling modules to those used by neuronal cells., (Copyright © 2011 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.)

Older persons with Mild Cognitive Impairment (MCI) feature neurobiological Alzheimer's Disease (AD) in 50% to 70% of the cases and develop dementia within the next 5 to 7 years. Current evidence suggests that biochemical, neuroimaging, electrophysiological, and neuropsychological markers can track the disease over time since the MCI stage (also called prodromal AD). The amount of evidence supporting their validity is of variable strength. We have reviewed the current literature and categorized evidence of validity into three classes: Class A, availability of multiple serial studies; Class B a single serial study or multiple cross sectional studies of patients with increasing disease severity from MCI to probable AD; and class C, multiple cross sectional studies of patients in the dementia stage, not including the MCI stage. Several Class A studies suggest that episodic memory and semantic fluency are the most reliable neuropsychological markers of progression. Hippocampal atrophy, ventricular volume and whole brain atrophy are structural MRI markers with class A evidence. Resting-state fMRI and connectivity, and diffusion MR markers in the medial temporal white matter (parahippocampus and posterior cingulum) and hippocampus are promising but require further validation. Change in amyloid load in MCI patients warrant further investigations, e.g. over longer period of time, to assess its value as marker of disease progression. Several spectral markers of resting state EEG rhythms that might reflect neurodegenerative processes in the prodromal stage of AD (EEG power density, functional coupling, spectral coherence, and synchronization) suffer from lack of appropriately designed studies. Although serial studies on late event-related potentials (ERPs) in healthy elders or MCI patients are inconclusive, others tracking disease progression and effects of cholinesterase inhibiting drugs in AD, and cross-sectional including MCI or predicting development of AD offer preliminary evidence of validity as a marker of disease progression from the MCI stage. CSF Markers, such as Aβ 1-42, t-tau and p-tau are valuable markers which support the clinical diagnosis of Alzheimer's disease. However, these markers are not sensitive to disease progression and cannot be used to monitor the severity of Alzheimer's disease. For Isoprostane F2 some evidence exists that its increase correlates with the progression and the severity of AD.

A familial form of Creutzfeldt-Jakob disease (CJD) is linked to the D178N/V129 prion protein (PrP) mutation. Tg(CJD) mice expressing the mouse homolog of this mutant PrP synthesize a misfolded form of the mutant protein, which is aggregated and protease resistant. These mice develop clinical and pathological features reminiscent of CJD, including motor dysfunction, memory impairment, cerebral PrP deposition, and gliosis. Tg(CJD) mice also display electroencephalographic abnormalities and severe alterations of sleep-wake patterns strikingly similar to those seen in a human patient carrying the D178N/V129 mutation. Neurons in these mice show swelling of the endoplasmic reticulum (ER) with intracellular retention of mutant PrP, suggesting that ER dysfunction could contribute to the pathology. These results establish a transgenic animal model of a genetic prion disease recapitulating cognitive, motor, and neurophysiological abnormalities of the human disorder. Tg(CJD) mice have the potential for giving greater insight into the spectrum of neuronal dysfunction in prion diseases.

A major prion protein (PrP) mutant that forms amyloid fibrils in the diseased brain of patients with Gerstmann-Sträussler-Scheinker syndrome (GSS) is a fragment of 7 kDa spanning from residues 81-82 to 144-153 of PrP. Analysis of ionic membrane currents, recorded with a lipid bilayer technique, revealed that the wild-type fragment PrP(82-146) WT and the partially scrambled PrP(82-146) (127-146) SC are capable of forming heterogeneous ion channels that are similar to those channels formed with PrP(106-126). In contrast, PrP(82-146) peptides in which the region from residue 106 to 126 had been scrambled (SC) showed a reduction in interaction with lipid membranes and did not form channels. The PrP(82-146) WT- and PrP(82-146) (127-146) SC-formed cation channels with fast kinetics are Cu2+ sensitive and rifampicin (RIF) insensitive, whereas the time-dependent inactivating channels formed by these same peptides are both Cu2+ and RIF insensitive. The presence of RIF in the solution before the addition of PrP(82-146) WT or PrP(82-146) (127-146) SC affected their incorporation into the lipid bilayers. PrP(82-146) WT and PrP(82-146) (127-146) SC fast cation channels formed in the presence of RIF appeared in an electrically semisilent state or an inactivated state. Increasing [Cd2+]cis enhanced the incorporation of PrP(82-146) WT and PrP(82-146) (127-146) SC channels formed in the presence of RIF. We conclude that the major PrP mutant fragment in the diseased brain of GSS patients is prone to form channels in neuronal membranes, causing their dysfunction. We propose that Cd2+ may accentuate the neurotoxicity of this channel-forming PrP fragment by enhancing its incorporation into the membrane.

Cerebral deposits of beta-amyloid (betaA) are a major feature in Alzheimer's disease. betaA is derived from amyloid precursor protein (APP). APP is subject to N- and O-glycosylation and undergoes a series of proteolytic cleavages that lead to the release of betaA or of a non-amyloidogenic secreted form of APP (APPs). We used primary neuronal and glial cultures to investigate how cholesterol affects the production and secretion of APPs. Exposure to cholesterol for 2 h did not change the neuronal release of APPs; after 6 h APPs release was slightly lower, whereas 24 h of exposure decreased APPs in the medium by approx. 60%. The time courses were similar in astrocytes and microglia preparations. To verify whether the effect of cholesterol was a consequence of membrane rigidification we tested the activity of ganglioside GM1 and prion protein fragment PrP 106-126, which affect membrane fluidity similarly to cholesterol, on APPs secretion. Neither altered the production of APPs. APP mRNA and the total amount of APP in the cells were slightly decreased by cholesterol after 2 and 24 h respectively. Immunoblot analysis of APP associated with neuronal cells and astrocytes indicated that cholesterol progressively decreased the glycosylated forms of the protein; a similar tendency was noted in cells treated with brefeldin A and monensin, two substances that interfere with protein glycosylation. The cell-surface biotinylation method showed that in cholesterol-treated cells APP reached the plasma membrane. Our results indicate that cholesterol decreases the secretion of APPs by interfering with APP maturation and inhibiting glycosylation of the protein; although APP is inserted in the membrane it is not cleaved by alpha-secretase.

Somatostatin expression in trisomy 16 mouse neuronal cultures has been studied to investigate the effects of the presence of an extra copy of the pre-pro-somatostatin (ppSS) gene on mouse chromosome 16. The immunoreactivity for somatostatin (SS) was considered in mixed cultures of neurons and glia cells and in neuron-enriched cultures as well as that for neuropeptide Y, glutamic acid decarboxylase, and gamma-enolase immunoreactivity the genes of which are not present on mouse chromosome 16. ppSS and pre-pro-neuropeptide Y (ppNPY) mRNA expression was evaluated and SS immunoreactivity in neurons analyzed by a morphometrical study. The extra copy of the ppSS gene resulted in a significantly increased level of the transcript in trisomic cultures, whereas the expression of the other neuropeptides did not differ. The absence of glial cells in these cultures reduced the number of SS-positive neurons making their number comparable in the trisomic and control cultures. Thus, in spite of higher expression of the ppSS mRNA in trisomic cultures, the determination of this peptidergic phenotype was influenced by the presence of neuroglial cells.

A new method is presented for the quantification of cell viability based on densitometry with computerized image analysis. Neuronal cells were stained with crystal violet and densitometric analysis was performed with an IBAS 2.0 image analyzer (Kontron/ Zeiss), using specially implemented dedicated software which integrates the optical density of the culture in each well with the area covered by the stained cells. To test the reliability of the densitometric method cortical cells were plated at different concentrations (5 x 10(4)-10(6)/ml); the standard curve obtained by analysis of crystal violet staining showed a linear proportion between cell number and optical density signal. The validation and accuracy of the method were assessed and compared with other methods using rat cortical cells cultured in vitro for 10 days and exposed to kainic acid (250 microM) for 24 h. Neuronal viability was reduced by 40-50% and comparison with direct cell counting, MTT assay, and spectrophotometric analysis confirmed that the method is simple, quick, and reliable.

Centrally injected endotoxin induced high levels of interleukin (IL)-6 in serum, but the mechanisms of this induction and the signal conveying the information from the brain to the periphery are not yet known. To help characterize the pathway of centrally mediated induction of IL-6 in periphery, the cytokine levels were measured in rat serum and cerebrospinal fluid at different times after intracerebro-ventricular endotoxin (LPS, 2.5 micrograms/rat). In the same experiments, IL-6 and IL-1 beta messenger RNA (mRNA) expression, measured by Northern blot analysis, were evaluated in the periphery (adrenals, lymph nodes, and mononuclear cells) and brain (hypothalamus, hippocampus and striatum). In serum, IL-6 levels were highest after 2h; then they rapidly decreased. IL-6 mRNA showed the same time-course in adrenals and lymph nodes. The pattern in the central nervous system was different: in the cerebrospinal fluid, IL-6 was detectable starting from 2h, reaching a plateaux at 4-8h and remaining detectable until 16 h. IL-6 mRNA expression in the brain areas showed a similar time-course, reaching a maximum at 4-8 h. IL-1 beta mRNA induction started at the same time in brain and periphery, i.e. 1 h after LPS, but the maximal effect was reached at 2 h in mononuclear cells, adrenals, and lymph nodes, and at 8 h in brain regions. The results indicate that circulating IL-6 induced by central LPS is produced mainly peripherally and that synthesis of IL-6 and IL-1 beta are regulated differently in the brain and periphery.

The binding of [3H]hemicholinium ([3H]HCh-3) to sodium-dependent high-affinity choline uptake sites provides a useful neuroanatomical and functional marker of the cholinergic system. We examined the autoradiographic distribution of [3H]HCh-3 binding sites in the forebrain of young (4-6 months) and old (32 months) rats. There was a widespread reduction of [3H]HCh-3 binding site density in the aged rat brain. This loss presented regional differences with maximal reduction in the medial and posterior striatum (55%) and in the dentate gyrus (47%), in limbic areas such as basolateral amygdala, tubercle olfactorium and piriform cortex the autoradiographic signal was about 25-30% lower. In aged hippocampus and cerebral cortex the density of [3H]HCh-3 binding sites was about 40% lower, the difference between young and senescent animals being less evident in the medial septum and basal nucleus. No significant alterations were observed in interpeduncular nucleus from old rats. These data are in agreement with the functional results obtained by measuring other cholinergic parameters in the aged rat and confirm the vulnerability of cholinergic system during aging.

An immunohistochemical technique for simultaneously visualizing two different antigens has been used to investigate the presence of the acidic dipeptide, N-acetyl-aspartyl-glutamate (NAAG), in cholinergic, noradrenergic-adrenergic, and serotonergic neurons within CNS. The brain slices were processed sequentially with purified antisera against NAAG and then monoclonal antibody against choline acetyltransferase (ChAT), a marker for cholinergic neurons, or antiserum against dopamine-beta-hydroxylase (DBH), a marker of noradrenergic-adrenergic neurons, or antiserum against serotonin (5HT). Both antigens were revealed by the peroxidase reaction but with different chromogens, which are easily distinguishable. An intense double staining of NAAG-like immunoreactivity (NAAG-LI) and ChAT was observed in the motoneurons of the spinal cord as well as in the several motor components of cranial nerve nuclei including facial, ambiguus, and trigeminal nuclei. A partial colocalization of NAAG-LI and ChAT was evident in the perikarya of the basal forebrain cholinergic system, whereas cholinergic neurons of the medial septum exhibited only sporadic staining for NAAG-LI. A complete coexistence of NAAG-LI and DBH was observed in the locus coeruleus. Most of the other noradrenergic and adrenergic cell groups of the medulla region exhibited substantial co-localization with the exception of the A2 cell group, which was virtually devoid of NAAG-LI. In the dorsal raphe, only a low percentage of serotonergic neurons stained for NAAG-LI. The co-existence of NAAG-LI and serotonin was more evident in the neurons of the median raphe, although the majority of cells failed to show double staining.(ABSTRACT TRUNCATED AT 250 WORDS)