"Back to Braak": Role of Nucleus Reuniens and Subcortical Pathways in Alzheimer's Disease Progression.

BACKGROUND: Patients with Alzheimer's Disease (AD) exhibit structural alterations of the thalamus that correlate with clinical symptoms. However, given the anatomical complexity of this brain structure, it is still unclear whether atrophy affects specific thalamic nuclei and modulates the clinical progression from a prodromal stage, known as Mild Cognitive Impairment (MCI), to full-fledged AD. OBJECTIVES: To characterize the structural integrity of distinct thalamic nuclei across the AD spectrum, testing whether MCI patients who convert to AD (c-MCI) show a distinctive pattern of thalamic structural alterations compared to patients who remain stable (s-MCI). DESIGN: Investigating between-group differences in the volumetric features of distinct thalamic nuclei across the AD spectrum. SETTING: Prodromal and clinical stages of AD. PARTICIPANTS: We analyzed data from 84 healthy control subjects (HC), 58 individuals with MCI, and 102 AD patients. The dataset was obtained from the AD Neuroimaging Initiative (ADNI-3) database. The MCI group was further divided into two subgroups depending on whether patients remained stable (s-MCI, n=22) or progressed to AD (s-MCI, n=36) in the 48 months following the diagnosis. MEASUREMENTS: A multivariate analysis of variance (MANOVA) assessed group differences in the volumetric features of distinct thalamic nuclei obtained from magnetic resonance (MR) images. A stepwise discriminant function analysis identified which feature most effectively predicted the conversion to AD. The corresponding predictive performance was evaluated through a Receiver Operating Characteristic approach. RESULTS: AD and c-MCI patients showed generalized atrophy of thalamic nuclei compared to HC. In contrast, no significant structural differences were observed between s-MCI and HC subjects. Compared to s-MCI, c-MCI individuals displayed significant atrophy of the nucleus reuniens and a trend toward significant atrophy in the anteroventral and laterodorsal nuclei. The discriminant function analysis confirmed the nucleus reuniens as a significant predictor of AD conversion, with a sensitivity of 0.73 and a specificity of 0.69. CONCLUSIONS: In line with the pathophysiological relevance of the nucleus reuniens proposed by seminal post-mortem studies on patients with AD, we confirm the pivotal role of this nucleus as a critical hub in the clinical progression to AD. We also propose a theoretical model to explain the evolving dysfunction of subcortical brain networks in the disease process.

Ascertainment bias in dementias: a secondary to tertiary centre analysis in Central Italy and conceptual review.

BACKGROUND AND AIMS: Ascertainment bias (AB) indicates a bias of an evaluation centre in estimating the prevalence/incidence of a disease due to the specific expertise of the centre. The aim of our study was to evaluate classification of different types of dementia in new cases appearing in secondary and tertiary centres, in order to evidence possible occurrence of AB in the various (secondary to tertiary) dementia centres. METHODS: To assess the mechanism of AB, the rates of new cases of the different forms of dementia reported by different centres were compared. The centres involved in the study were 11 hospital-based centres including a tertiary centre, located in the University Department of Clinical Neurology. The tertiary centre is endowed with state-of-the-art diagnostic facilities and its scientific production is prominently focused on dementia with Lewy bodies (DLB) thus suggesting the possible occurrence of a bias. Four main categories of dementia were identified: Alzheimer's disease (AD), DLB, fronto-temporal dementia (FTD), vascular dementia (VaD), with other forms in a category apart. The classification rate of new cases of dementia in the tertiary centre was compared with rates reported by secondary centres and rates of recoding were calculated during a follow-up of 2 years. RESULTS: The study classified 2,042 newly diagnosed cases of dementia in a population of 1,370,000 inhabitants of which 315,000 were older than 65. AD was categorized in 48-52 % of cases, DLB in 25-28 %, FTD in 2-4 % and VaD in 17-28 %. During the 2-year follow-up the diagnosis was re-classified in 40 patients (3 %). The rate of recoding was 5 % in the tertiary centre, 2-8 % in referrals from secondary to tertiary centre, 2-10 % in recodings performed in secondary centres and addressed to tertiary centre. Recoding or percentages of new cases of AD or DLB were not different in the comparison between secondary or between secondary and tertiary centres. FTD and VaD were instead significantly recoded. CONCLUSION: The results of the study suggest that in a homogeneous area, AB is not interfering with diagnosis of AD or DLB.

Alemtuzumab treatment of multiple sclerosis in real-world clinical practice: A report from a single Italian center.

BACKGROUND: Alemtuzumab, is a compound approved for highly active MS, and, in Europe, employed after the use of other disease-modifying treatments (DMTs) with an escalation approach or used as a first therapeutic option. The occurrence of secondary autoimmune adverse events and or infections can differ depending on the employed approach. OBJECTIVE: To evaluate the efficacy and safety of alemtuzumab in real-world MS population that encompassed patients previously treated with other DMTs. METHODS: 35 patients, treated with alemtuzumab in a single MS Center, were followed for at least 36 months. The study investigated the prevalence of patients reaching the phase of the non-active disease (NEDA-3). All the adverse events were also reported, and correlations assessed. RESULTS: At the 36-month follow-up, 66,7% of patients achieved the NEDA-3 status, 90,5% of the patients were relapse-free, 85,7% showed no signs of disability progression, nor signs of MRI activity. Adverse events were observed in 45,7% of the patients and ranked as severe in 23% of them. Cases of autoimmune hemolytic anemia (AIHA), pancytopenia, viral hepatitis E, and noninfectious meningo-encephalomyelitis were found and reported. For these complications, the post hoc analysis showed possible interactive factors and causality related to previous DMT treatments. CONCLUSIONS: In a real-world MS population like the one investigated in our study, alemtuzumab was found to be an effective treatment when employed as an escalation or rescue therapy. The compound exhibits a variable safety profile and frequent adverse events that are likely depending on previous treatments and their impact on the immune system.

Mediation of neuronal apoptosis by enhancement of outward potassium current.

Apoptosis of mouse neocortical neurons induced by serum deprivation or by staurosporine was associated with an early enhancement of delayed rectifier (IK) current and loss of total intracellular K+. This IK augmentation was not seen in neurons undergoing excitotoxic necrosis or in older neurons resistant to staurosporine-induced apoptosis. Attenuating outward K+ current with tetraethylammonium or elevated extracellular K+, but not blockers of Ca2+, Cl-, or other K+ channels, reduced apoptosis, even if associated increases in intracellular Ca2+ concentration were prevented. Furthermore, exposure to the K+ ionophore valinomycin or the K+-channel opener cromakalim induced apoptosis. Enhanced K+ efflux may mediate certain forms of neuronal apoptosis.

Survey of domestic accidents in the elderly in the Province of Genoa (northern Italy).

INTRODUCTION: Accidents in the home are a major public health issue in most industrialised countries, as they are a frequent cause of injury and death. Moreover, since a considerable portion of such accidents involve elderly people, it is important to assess their social impact in this population. In Italy, the available data indicate that well over 3 million people per year suffer accidents in the home, and that this number is rising. The aim of the present study was to evaluate the number, characteristics and causes of domestic accidents among the elderly. MATERIALS AND METHODS: The study population was made up of subjects of both sexes aged between 65 and 92 years admitted to first aid units and emergency departments of hospitals in Genoa. The investigation was conducted by means of an ad hoc questionnaire designed to record the circumstances of the accident, the functional capacity of the subject involved, any risky behaviour enacted and the safety profile of the subject's home. RESULTS: The study enrolled 111 voluntary participants: 62 women and 49 men. At the time of the accident, subjects were engaged in the following activities: housework (36.9%) "rest" (14.5%), ablutions (10%), gardening (9%), leisure activities (8.1%), eating and drinking (2.7%). The most common injuries were bruises (39.6%), followed by fractures (23.4%) and cuts (23.4%); the frequency of other, some time more severe injures (burns, poisoning, asphyxia, crush injuries, etc.) was,fortunately, very low. Anyway, taking into account their consequences, their surveillance and prevention is very important. Most subjects were deemed to be in good health and, in 76% of cases, the safety profile of their houses proved to be satisfactory. DISCUSSION AND CONCLUSIONS: The data collected during this survey are in line with those yielded by national and international studies. They show that the elderly are very vulnerable to domestic accidents and that, even in the event of only slight injury, the management of elderly victims requires a strong organisational commitment on the part of relatives and considerable financial resources for healthcare services. Falling proved to be the main cause of injury. Clearly, efforts to reduce the cost of accidents in the home should aim to implement preventive intervention among elderly people, since the elderly population is destined to grow as a result of increasing life expectancy. In particular, preventive action should focus on reducing the incidence of falls by eliminating risk-related structural features in domestic settings as far as possible and by raising public awareness of the problem through health education campaigns.

Ca2+-Zn2+ permeable AMPA or kainate receptors: possible key factors in selective neurodegeneration.

Neurological diseases, including global ischemia, Alzheimer's disease and amyotrophic lateral sclerosis, are characterized by selective patterns of neurodegeneration. Most studies of potential glutamate-receptor-mediated contributions to disease have focused on the highly Ca2+-permeable and widely distributed NMDA-receptor channel. However, an alternative hypothesis is that the presence of AMPA- or kainate-receptor channels that are directly permeable to Ca2+ ions (Ca-A/K-receptor channels) is of greater significance to the neuronal loss seen in these conditions. Besides a restricted distribution and high Ca2+ permeability, two other factors make Ca-A/K receptors appealing candidate contributors to selective injury: their high permeability to Zn2+ ions and the possibility that their numbers increase in disease-associated conditions. Further characterization of the functions of these channels should result in new approaches to treatment of these conditions.

Altered Kv2.1 functioning promotes increased excitability in hippocampal neurons of an Alzheimer's disease mouse model.

Altered neuronal excitability is emerging as an important feature in Alzheimer's disease (AD). Kv2.1 potassium channels are important modulators of neuronal excitability and synaptic activity. We investigated Kv2.1 currents and its relation to the intrinsic synaptic activity of hippocampal neurons from 3xTg-AD (triple transgenic mouse model of Alzheimer's disease) mice, a widely employed preclinical AD model. Synaptic activity was also investigated by analyzing spontaneous [Ca(2+)]i spikes. Compared with wild-type (Non-Tg (non-transgenic mouse model)) cultures, 3xTg-AD neurons showed enhanced spike frequency and decreased intensity. Compared with Non-Tg cultures, 3xTg-AD hippocampal neurons revealed reduced Kv2.1-dependent Ik current densities as well as normalized conductances. 3xTg-AD cultures also exhibited an overall decrease in the number of functional Kv2.1 channels. Immunofluorescence assay revealed an increase in Kv2.1 channel oligomerization, a condition associated with blockade of channel function. In Non-Tg neurons, pharmacological blockade of Kv2.1 channels reproduced the altered pattern found in the 3xTg-AD cultures. Moreover, compared with untreated sister cultures, pharmacological inhibition of Kv2.1 in 3xTg-AD neurons did not produce any significant modification in Ik current densities. Reactive oxygen species (ROS) promote Kv2.1 oligomerization, thereby acting as negative modulator of the channel activity. Glutamate receptor activation produced higher ROS levels in hippocampal 3xTg-AD cultures compared with Non-Tg neurons. Antioxidant treatment with N-Acetyl-Cysteine was found to rescue Kv2.1-dependent currents and decreased spontaneous hyperexcitability in 3xTg-AD neurons. Analogous results regarding spontaneous synaptic activity were observed in neuronal cultures treated with the antioxidant 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox). Our study indicates that AD-related mutations may promote enhanced ROS generation, oxidative-dependent oligomerization, and loss of function of Kv2.1 channels. These processes can be part on the increased neuronal excitability of these neurons. These steps may set a deleterious vicious circle that eventually helps to promote excitotoxic damage found in the AD brain.

AMPA exposures induce mitochondrial Ca(2+) overload and ROS generation in spinal motor neurons in vitro.

The reason for the selective vulnerability of motor neurons in amyotrophic lateral sclerosis (ALS) is primarily unknown. A possible factor is the expression by motor neurons of Ca(2+)-permeable AMPA/kainate channels, which may permit rapid Ca(2+) influx in response to synaptic receptor activation. However, other subpopulations of central neurons, most notably forebrain GABAergic interneurons, consistently express large numbers of these channels but do not degenerate in ALS. Indeed, when subjected to identical excitotoxic exposures, motor neurons were more susceptible than GABAergic neurons to AMPA/kainate receptor-mediated neurotoxicity. Microfluorimetric studies were performed to examine the basis for the difference in vulnerability. First, AMPA or kainate exposures appeared to trigger substantial mitochondrial Ca(2+) loading in motor neurons, as indicated by a sharp increase in intracellular Ca(2+) after addition of the mitochondrial uncoupler carbonyl cyanide p-(trifluoromethoxy)phenyl hydrazone (FCCP) after the agonist exposure. The same exposures caused little mitochondrial Ca(2+) accumulation in GABAergic cortical neurons. Subsequent experiments examined other measures of mitochondrial function to compare sequelae of AMPA/kainate receptor activation between these populations. Brief exposure to either AMPA or kainate caused mitochondrial depolarization, assessed using tetramethylrhodamine ethylester, and reactive oxygen species (ROS) generation, assessed using hydroethidine, in motor neurons. However, these effects were only seen in the GABAergic neurons after exposure to the nondesensitizing AMPA receptor agonist kainate. Finally, addition of either antioxidants or toxins (FCCP or CN(-)) that block mitochondrial Ca(2+) uptake attenuated AMPA/kainate receptor-mediated motor neuron injury, suggesting that the mitochondrial Ca(2+) uptake and consequent ROS generation are central to the injury process.

Zn(2+): a novel ionic mediator of neural injury in brain disease.

Zn(2+) is the second most prevalent trace element in the body and is present in particularly large concentrations in the mammalian brain. Although Zn(2+) is a cofactor for many enzymes in all tissues, a unique feature of brain Zn(2+) is its vesicular localization in presynaptic terminals, where its release is dependent on neural activity. Although the physiological significance of synaptic Zn(2+) release is little understood, it probably plays a modulatory role in synaptic transmission. Furthermore, several lines of evidence support the idea that, upon excessive synaptic Zn(2+) release, its accumulation in postsynaptic neurons contributes to the selective neuronal loss that is associated with certain acute conditions, including epilepsy and transient global ischaemia. More speculatively, Zn(2+) dis-homeostasis might also contribute to some degenerative conditions, including Alzheimer's disease. Further elucidation of the pathological actions of Zn(2+) in the brain should result in new therapeutic approaches to these conditions.

Rethinking the excitotoxic ionic milieu: the emerging role of Zn(2+) in ischemic neuronal injury.

Zn(2+) plays an important role in diverse physiological processes, but when released in excess amounts it is potently neurotoxic. In vivo trans-synaptic movement and subsequent post-synaptic accumulation of intracellular Zn(2+) contributes to the neuronal injury observed in some forms of cerebral ischemia. Zn(2+) may enter neurons through NMDA channels, voltage-sensitive calcium channels, Ca(2+)-permeable AMPA/kainate (Ca-A/K) channels, or Zn(2+)-sensitive membrane transporters. Furthermore, Zn(2+) is also released from intracellular sites such as metallothioneins and mitochondria. The mechanisms by which Zn(2+) exerts its potent neurotoxic effects involve many signaling pathways, including mitochondrial and extra-mitochondrial generation of reactive oxygen species (ROS) and disruption of metabolic enzyme activity, ultimately leading to activation of apoptotic and/or necrotic processes. As is the case with Ca(2+), neuronal mitochondria take up Zn(2+) as a way of modulating cellular Zn(2+) homeostasis. However, excessive mitochondrial Zn(2+) sequestration leads to a marked dysfunction of these organelles, characterized by prolonged ROS generation. Intriguingly, in direct comparison to Ca(2+), Zn(2+) appears to induce these changes with a considerably greater degree of potency. These effects are particularly evident upon large (i.e., micromolar) rises in intracellular Zn(2+) concentration ([Zn(2+)](i)), and likely hasten necrotic neuronal death. In contrast, sub-micromolar [Zn(2+)](i) increases promote release of pro-apoptotic factors, suggesting that different intensities of [Zn(2+)](i) load may activate distinct pathways of injury. Finally, Zn(2+) homeostasis seems particularly sensitive to the environmental changes observed in ischemia, such as acidosis and oxidative stress, indicating that alterations in [Zn(2+)](i) may play a very significant role in the development of ischemic neuronal damage.

Measuring zinc in living cells. A new generation of sensitive and selective fluorescent probes.

New fluorescent indicators with nanomolar to micromolar affinities for Zn(2+) have been synthesized in wavelengths from UV to the far red. The UV light-excited indicators are ratiometric. The visible wavelength indicators are non-ratiometric and exhibit large and pH-independent fluorescence increases with increasing zinc concentrations, with little to no sensitivity to physiologically relevant Ca(2+) concentrations. Experiments in neuronal and non-neuronal cell cultures show the new indicators to retain their sensitivity to and selectivity for zinc after conversion to cell-permeable forms.

Circadian rhythm of insulin-induced hypoglycemia in man.

Four healthy men were given an insulin tolerance test (0.1 IU/kg BW) six times in different days at the following clock hours: 0400 h, 0800h, 1200 h, 1600 h, 2000 h, and 2400 h, in random order. Four days separated two consecutive tests. The glucose disappearance rate (K value) was calculated for each test between 5 min and 25 min after iv injection of insulin. The statistical evaluation by Cosinor test showed a statistically significant circadian rhythm for the hypoglycemic action of exogenous insulin with a peak at 1030 h (95% condifence limits: 0830 to 1430 h).

Dendritic localization of Ca(2+)-permeable AMPA/kainate channels in hippocampal pyramidal neurons.

Although it is well established that cortical and hippocampal gamma-aminobutyric acid (GABA)-ergic neurons generally have large numbers of Ca(2+)-permeable alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate channels (Ca-A/K channels), their presence on pyramidal neurons is controversial. Ca2+ permeability of AMPA channels is regulated by expression of a particular glutamate receptor subunit (GluR2), which confers Ca2+ impermeability to heteromeric channels. Most electrophysiology studies, as well as in situ hybridization and immunolabeling studies demonstrating expression of GluR2 mRNA or peptide in pyramidal neurons, have provided evidence against the presence of Ca-A/K channels on pyramidal neurons. However, observations that pyramidal neurons often appear to be labeled by kainate-stimulated Co2+ influx (Co2+(+) cells), a histochemical stain that identifies cells possessing Ca-A/K channels, suggests that they may have these channels. The present study futher examines cellular and subcellular distribution of Ca-A/K channels on hippocampal pyramidal neurons in slice as well as in culture. To this end, techniques of kainate-stimulated Co2+ influx labeling, supplemented by AMPA receptor subunit immunocytochemistry and fluorescent imaging of kainate-stimulated intracellular Ca2+ ([Ca2+]i) rises are employed. Co2+ labeling is often seen in pyramidal neuronal dendrites in both slice and in culture. In addition, although GluR1 and 4 staining in these neurons is often seen in the soma and dendrites, GluR2 label, when evident, is generally more restricted to the soma. Finally, measurement of kainate-stimulated [Ca2+]i rises in cultured neurons, assessed by using low affinity Ca2+ indicators in the presence of N-methyl-D-aspartate (NMDA) receptor and voltage-sensitive Ca2+ channel blockade, often shows dendritic rises to precede those in the somata. Thus, these data support the hypothesis that Ca-A/K channels are present in dendritic domains of many pyramidal neurons, and may help to provide resolution of the apparently conflicting data regarding their distribution.

Nitric oxide reduces Ca(2+) and Zn(2+) influx through voltage-gated Ca(2+) channels and reduces Zn(2+) neurotoxicity.

The translocation of synaptic Zn(2+) from nerve terminals into selectively vulnerable neurons may contribute to the death of these neurons after global ischemia. We hypothesized that cellular Zn(2+) overload might be lethal for reasons similar to cellular Ca(2+) overload and tested the hypothesis that Zn(2+) neurotoxicity might be mediated by the activation of nitric oxide synthase. Although Zn(2+) (30-300microM) altered nitric oxide synthase activity in cerebellar extracts in solution, it did not affect nitric oxide synthase activity in cultured murine neocortical neurons. Cultured neurons exposed to 300-500microM Zn(2+) for 5min under depolarizing conditions developed widespread degeneration over the next 24h that was unaffected by the concurrent addition of the nitric oxide synthase inhibitor N(G)-nitro-L-arginine. Furthermore, Zn(2+) neurotoxicity was attenuated when nitric oxide synthase activity in the cultures was induced by exposure to cytokines, exogenous nitric oxide was added or nitric oxide production was pharmacologically enhanced. The unexpected protective effect of nitric oxide against Zn(2+) toxicity may be explained, at least in part, by reduction of toxic Zn(2+) entry. Exposure to nitric oxide donors reduced Ba(2+) current through high-voltage activated calcium channels, as well as K(+)-stimulated neuronal uptake of 45Ca(2+) or 65Zn(2+). The oxidizing agents thimerosal and 2,2'-dithiodipyridine also reduced K(+)-stimulated cellular 45Ca(2+) uptake, while akylation of thiols by pretreatment with N-ethylmaleimide blocked the reduction of 45Ca(2+) uptake by a nitric oxide donor.The results suggest that Zn(2+)-induced neuronal death is not mediated by the activation of nitric oxide synthase; rather, available nitric oxide may attenuate Zn(2+) neurotoxicity by reducing Zn(2+) entry through voltage-gated Ca(2+) channels, perhaps by oxidizing key thiol groups.

Calcium ionophores can induce either apoptosis or necrosis in cultured cortical neurons.

Cultured cortical neurons exposed for 24 h to low concentrations of the Ca2+ ionophores, ionomycin (250 nM) or A-23187 (100 nM), underwent apoptosis, accompanied by early degeneration of neurites, cell body shrinkage, chromatin condensation and internucleosomal DNA fragmentation. This death could be blocked by protein synthesis inhibitors, as well as by the growth factors brain-derived neurotrophic factor or insulin-like growth factor I. If the ionomycin concentration was increased to 1-3 microM, then neurons underwent necrosis, accompanied by early cell body swelling without DNA laddering, or sensitivity to cycloheximide or growth factors. Calcium imaging with Fura-2 suggested a possible basis for the differential effects of low and high concentrations of ionomycin. At low concentrations, ionomycin induced greater increases in intracellular Ca2+ concentration in neurites than in neuronal cell bodies, whereas at high concentrations, ionomycin produced large increases in intracellular Ca2+ concentration in both neurites and cell bodies. We hypothesize that the ability of low concentrations of Ca2+ ionophores to raise intracellular Ca2+ concentration preferentially in neurites caused early neurite degeneration, leading to loss of growth factor availability to the cell body and consequent apoptosis, whereas high concentrations of ionophores produced global cellular Ca2+ overload and consequent necrosis.

Glutamate triggers preferential Zn2+ flux through Ca2+ permeable AMPA channels and consequent ROS production.

ZN2+ co-released with glutamate at excitatory synaptic sites can enter and cause injury to postsynaptic neurons. While prior studies using the slowly desensitizing agonist kainate suggested preferential Zn2+ permeation through Ca2+ permeable AMPA/kainate (Ca-A/K) channels, the present study aims to assess relevance of those findings upon more physiological receptor activation. Microfluorimetric techniques were used to measure [Zn2+]i attained upon exposure to the rapidly desensitizing agonist AMPA or to the physiological agonist glutamate, in the presence of 300 microM Zn2+. Under these conditions, micromolar [Zn2+]i rises (delta[Zn2+]i) were still observed to occur selectively in the subset of neurons that express large numbers of Ca-A/ K channels. Further studies using the oxidation sensitive dye, hydroethidine, revealed Zn2+-dependent reactive oxygen species generation that paralleled delta[Zn2+]i, with rapid oxidation only observed in the case of Zn2+ entry through Ca-A/K channels.

3-Nitropropionic acid induces apoptosis in cultured striatal and cortical neurons.

Ingestion of 3-nitropropionic acid (3-NPA) in moldy sugar cane causes brain damage in children. The mechanism of 3-NPA toxicity is thought to be inhibition of energy production, leading to ATP depletion and excitotoxicity. We exposed cultured mouse striatal or cortical neurons to 1-2 mM 3-NPA for 48 h. This exposure produced gradual neuronal degeneration characterized by cell body shrinkage and DNA fragmentation. Addition of glutamate antagonists during 3-NPA exposure did not reduce neuronal death. However, addition of the macromolecular synthesis inhibitors cycloheximide, emetine or actinomycin D markedly reduced neuronal death. Our results do not exclude that 3-NPA can induce excitotoxicity in more intact systems, but raise the additional possibility that 3-NPA may also act to induce neuronal apoptosis.