Generation and Characterization of the First Murine Model of Alzheimer's Disease with Mutated AßPP Inserted in a BALB/c Background (C.B6/J-APPswe).

BACKGROUND: Numerous mouse models of Alzheimer's disease (AD) are available, but all suffer from certain limitations, thus prompting further attempts. To date, no one model exists with amyloidopathy in a BALB/c strain. OBJECTIVE: To generate and characterize the C.B6/J-APPswe mouse, a model of AD with a mutated human gene for the amyloid-ß protein precursor (AßPP) inserted in a BALB/c background. METHODS: We analyzed five groups at different ages (3, 6, 9, 12, and 16-18 months) of C.B6/J-APPswe and wild-type mice (50% males and 50% females) for the main hallmarks of AD by western blotting, amyloid-ß (Aß) ELISA, immunocytochemistry, electrophysiology, and behavioral tests. RESULTS: The C.B6/J-APPswe mouse displays early AßPP and Aß production, late amyloid plaques formation, high level of Tau phosphorylation, synaptic deficits (reduced density and functional impairment due to a reduced post-synaptic responsiveness), neurodegeneration caused by apoptosis and necroptosis/necrosis, microgliosis, astrocytic abnormalities, and sex-related differences in explorative behavior, anxiety-like behavior, and spatial long-term and working memories. Social housing is feasible despite the intra-cage aggressiveness of male animals. CONCLUSION: C.B6/J-APPswe mice develop most of the distinctive features of AD and is a suitable model for the study of brain atrophy mechanisms and of the differences between males and females in the onset of cognitive/non-cognitive deficits.

The effect of astaxanthin on the aging rat brain: gender-related differences in modulating inflammation.

BACKGROUND: Astaxanthin (Ax) is a ketocarotenoid of the xanthophyll family with activities such as antioxidation, preservation of the integrity of cell membranes and protection of the redox state and functional integrity of mitochondria. The aim of this study was to investigate potential gender-related differences in the effect of Ax on the aging rat brain. RESULTS: In females, interleukin 1 beta (IL1ß) was significantly lower in treated rats in both cerebral areas, and in the cerebellum, treated animals also had significantly higher IL10. In males, no differences were found in the cerebellum, but in the hippocampus, IL1ß and IL10 were significantly higher in treated rats. CONCLUSION: These are the first results to show gender-related differences in the effect of Ax on the aging brain, emphasizing the necessity to carefully analyze female and male peculiarities when the anti-aging potentialities of this ketocarotenoid are evaluated. The observations lead to the hypothesis that Ax exerts different anti-inflammatory effects in female and male brains.

Effect of cognitive training on the expression of brain-derived neurotrophic factor in lymphocytes of mild cognitive impairment patients.

To identify biomarkers associated with cognitive stimulation of mild cognitive impairment (MCI) patients, we performed quantitative real-time reverse transcriptase polymerase chain reaction for brain-derived neurotrophic factor (BDNF) mRNA in peripheral lymphocytes of MCI and healthy subjects undergoing a multi-component cognitive training (CT) program. CT determined a significant decrease of BDNF mRNA levels in MCI (fold change=0.31) as compared to healthy subjects (fold change=0.86). It has been reported that in MCI there is an increase of BDNF serum levels, and our findings could indicate a positive effect of CT in restoring pre-disease levels of expression.

Early selective vulnerability of synapses and synaptic mitochondria in the hippocampal CA1 region of the Tg2576 mouse model of Alzheimer's disease.

Increasing experimental evidence indicates that synaptic alterations play a key role in cognitive decline in Alzheimer's disease (AD). Functional and structural synaptic changes progressively take place, beginning in the early phase of AD, mainly triggered by intracellular accumulation of soluble amyloid-ß (Aß) oligomers. These peptides also accumulate within mitochondria, heavily affecting their function and morphology, particularly in synaptic compartments. To better understand the role of mitochondrial impairment in synaptic alterations during the early stages of AD, a morphological investigation was performed by means of electron microscopy in the hippocampus of 3 month-old Tg2576 and transgene-negative littermate mice. In the stratum moleculare of CA1 pyramidal cells (SMCA1) of transgenic animals compared to controls, we found significantly larger and less numerous synapses, with a significantly reduced fraction of the perforated subtype, as well as significantly smaller and more numerous mitochondria. In contrast, no differences between the two groups of mice were found in the inner molecular layer of the dentate gyrus. The reduction of synaptic contacts in SMCA1 indicates a precocious vulnerability of this region, and the synaptic enlargement may reflect a compensating process aimed at maintaining the overall contact density. Accordingly, mitochondrial modifications may represent a plastic reactive phenomenon aimed at sustaining the increased energy needs for synaptic remodeling, since mitochondrial morphology was perfectly preserved and smaller mitochondria are metabolically more efficient. Thus, morphological changes occurring at synaptic level in SMCA1 of 3 month-old Tg2576 mice might reflect a precocious vulnerability associated with a residual plastic reactivity which may slow down functional alterations.

Platelets in Alzheimer's disease-associated cellular senescence and inflammation.

Alzheimer's disease (AD) is a complex degenerative disorder of the brain, associated with a progressive cognitive decline. Age is the main risk factor with almost half of the population above 90 years affected by this pathology. AD and brain aging share common molecular changes, so it has been hypothesized that AD could be a form of accelerated brain aging. In this context, senescenceassociated mechanisms could be a valuable target of investigation both to analyze the causes of this disease and to define therapeutic strategies. Senescent phenotypes of glia and neurons, as well as of peripheral cells, have been described in AD. Much evidence indicate that vascular impairment is a fundamental contributor to AD pathology and platelets are generally considered a key element because they represent the link between amyloid-ß (Aß) deposition, peripheral inflammation and endothelial senescence. Both activated and senescent platelets are a source of Aß, in addition activated platelets secrete many proinflammatory mediators that could contribute to increased peripheral inflammation and endothelial senescence. Treatments aimed to target peripheral endothelial senescence include antioxidants and some substances, such as aspirin, that modulate platelet aggregation and inflammatory response. Heparin has been proposed as a treatment for senile dementia and exhibits anti-inflammatory action as well as inhibitory effects on Aß assembly. Identifying peripheral targets for AD treatments could also result advantageous as it would be possible to monitor directly their efficacy. Nevertheless more research is needed to clarify all the different aspects and interactions of blood cells, vascular cells and their secretory products.

Peripheral inflammatory biomarkers of Alzheimer's disease: the role of platelets.

Alzheimer's disease is an age-dependent neurodegenerative disorder characterized by loss of neurons, synaptic degeneration, senile plaques and neurofibrillary tangles. Besides these hallmarks, increased accumulation of activated microglia, astrocytes and leukocytes adhering to postcapillary venules are observed in the affected brain areas, suggesting the presence of an ongoing inflammatory process. As neuroinflammation triggers the activation of peripheral immune system, many studies have analyzed circulating inflammatory biomarkers, including basal or stimulated levels of cytokines and related molecules in blood of Alzheimer's patients, but with conflicting results. Platelets are an important source of amyloid-ss (Ass) in the circulatory system and play an important pro-inflammatory role. Upon activation, they adhere to leukocytes and endothelial cells by means of adhesive proteins like P-selectin, platelet endothelial cell adhesion molecule-1 (PECAM) and intercellular adhesion molecule-1 and -2 (ICAM-1 and -2) and secrete inflammatory mediators (chemokines, interleukins). In addition, platelets contain important enzymes involved in inflammatory intermediary synthesis like phospholipase A(2) (PLA(2)) and cyclooxygenase-2 (COX-2), and recent reports demonstrated significant changes in platelet levels and activities in Alzheimer's disease. Thus, as platelets represent an important link between Ass deposition and inflammatory reactions especially at endothelial level, they can be considered a valuable cellular model to evaluate potential peripheral inflammatory biomarkers in Alzheimer's disease.

A ketogenic diet increases succinic dehydrogenase (SDH) activity and recovers age-related decrease in numeric density of SDH-positive mitochondria in cerebellar Purkinje cells of late-adult rats.

Ketogenic diets (KDs) have been applied in the therapy of paediatric epilepsy for nearly a century. Recently, beneficial results have also been reported on metabolic disorders and neurodegeneration, designating aged individuals as possible recipients. However, KDs efficacy decrease after the suckling period, and very little is known about their impact on the aging brain. In the present study, the effect on the neuronal energetic supply of a KD containing 20% of medium chain triglycerides (MCT) was investigated in Purkinje cells of the cerebellar vermis of late-adult (19-month-old) rats. The animals were fed with the KD for 8 weeks, and succinic dehydrogenase (SDH) activity was cytochemically determined. The following parameters of SDH-positive mitochondria were evaluated by the use of a computer-assisted image analysis system connected to a transmission electron microscope: numeric density (Nv), average volume (V), volume density (Vv), and cytochemical precipitate area/mitochondrial area (R). Young, age-matched, and old animals fed with a standard chow were used as controls. We found significantly higher Nv in MCT-KD-fed rats vs. all the control groups, in young vs. late-adult and old controls, and in late-adult vs. old controls. V and Vv showed no significant differences among the groups. R was significantly higher in MCT-KD-fed rats vs. all the control animals, and in old vs. young and late-adult controls. Present data indicate that the ketogenic treatment counteracted age-related decrease in numeric density of SDH-positive mitochondria, and enhanced their metabolic efficiency. Given the central role of mitochondrial impairment in age-related physio-pathological changes of the brain, these findings may represent a starting point to examine novel potentialities for KDs.

A ketogenic diet increases succinic dehydrogenase activity in aging cardiomyocytes.

Impairment of energy metabolism and an increase of reactive oxygen species (ROS) production seem to play a major role in age-related apoptotic loss of cardiomyocytes. Succinic dehydrogenase (SDH) is an important marker of the mitochondrial capability to provide an adequate amount of ATP. Moreover, because of its unique redox properties, SDH activity contributes to maintain the reduced state of the ubiquinone pool. Recent reports have shown that ketone body intake improves cardiac metabolic efficiency and exerts a cardioprotective antioxidant action, we therefore performed a cytochemical investigation of SDH activity in cardiomyocytes of late-adult (19-month-old) rats fed for 8 weeks with a medium-chain triglycerides ketogenic diet (MCT-KD). Young, age-matched and old animals fed with a standard chow were used as controls. The overall area of the precipitates (PA) from SDH activity and the area of the SDH-positive mitochondria (MA) were measured. The percent ratios PA/MA and MA/total myocardial tissue area (MA/TA) were the parameters taken into account. We found that PA/MA was significantly higher in young control rats and in MCT-KD-fed rats versus late-adult and old control rats and in young control versus MCT-KD-fed rats. MA/TA of MCT-KD-fed rats was significantly higher versus age-matched and old control rats and tended to be higher versus young control rats; this parameter was significantly higher in young versus old control rats. Thus, MCT-KD intake partially recovers age-related decrease of SDH activity and increases the myocardial area occupied by metabolically active mitochondria. These effects might counteract metabolic alterations leading to apoptosis-induced myocardial atrophy and failure during aging.

Ketogenic diets cause opposing changes in synaptic morphology in CA1 hippocampus and dentate gyrus of late-adult rats.

Ketogenic diets (KDs) have beneficial effects on several diseases, such as epilepsy, mitochondriopathies, cancer, and neurodegeneration. However, little is known about their effects on aging individuals. In the present study, late-adult (19-month-old) rats were fed for 8 weeks with two medium chain triglycerides (MCT)-KDs, and the following morphologic parameters reflecting synaptic plasticity were evaluated in stratum moleculare of hippocampal CA1 region (SM CA1) and outer molecular layer of hippocampal dentate gyrus (OML DG): average area (S), numeric density (Nv(s)), and surface density (Sv) of synapses, and average volume (V), numeric density (Nv(m)), and volume density (Vv) of synaptic mitochondria. In SM CA1, MCT-KDs induced the early appearance of the morphologic patterns typical of old animals (higher S and V, and lower Nv(s) and Nv(m)). On the contrary, in OML DG, Sv and Vv of MCT-KDs-fed rats were higher (as a result of higher Nv(s) and Nv(m)) versus controls; these modifications are known to improve synaptic function and metabolic supply. The opposite effects of MCT-KDs might reflect the different susceptibility to aging processes: OML DG is less vulnerable than SM CA1, and the reactivation of ketone bodies uptake and catabolism might occur more efficiently in this region, allowing the exploitation of their peculiar metabolic properties. Present findings provide the first evidence that MCT-KDs may cause opposite morphologic modifications, being potentially harmful for SM CA1 and potentially advantageous for OML DG. This implies risks but also promising potentialities for their therapeutic use during aging.

Reactive structural dynamics of synaptic mitochondria in ischemic delayed neuronal death.

The effect of transient global ischemia on the ultrastructural features of synaptic mitochondria at the distal dendrites of CA1 hippocampal neurons was investigated in 3-month-old rats. Sham surgery was performed on age-matched controls. The number of mitochondria/microm3 of neurophils (Nv: numeric density), the mitochondrial average size (average volume: V), and longer diameter (Fmax) as well as the overall fraction of neurophils occupied by mitochondria (volume density: Vv) were measured by computer-assisted morphometry. In ischemic rats, a 10% nonsignificant decrease of Nv was found, V increased nonsignificantly by 11%, and Fmax increased nonsignificantly by 5% versus controls. As a final outcome of these balanced changes, Vv remained unchanged between the two experimental groups investigated. In ischemic animals, the percentage distribution of V showed that the population of CA1 synaptic mitochondria was composed by an increased fraction of oversized organelles, while the Fmax distribution revealed that this enlargement was due to an increased percentage of elongated organelles. Thus, the observed increase in size should not be considered as a swelling phenomenon; on the contrary, it may represent a physiological and well-documented step in mitochondrial biogenesis. The above parameters are currently supposed to provide information on the adaptive structural reorganization of mitochondrial morphology under different environmental stimulations. Conceivably, these findings document a positive reactive response to ischemia of the mitochondrial structural dynamics at CA1 synaptic terminals and suggest consideration of these organelles as reliable targets in the development of neuroprotective therapeutic interventions to treat vascular brain diseases, for example, stroke.

Experimental apoptosis provides clues about the role of mitochondrial changes in neuronal death.

A quantitative morphometric study has been carried out in human neuroblastoma SK-N-BE cells to evaluate the ultrastructural features and the metabolic efficiency of mitochondria involved in the early steps of apoptosis. In mitochondria from control and apoptotic cells cytochrome oxidase (COX) activity was estimated by preferential cytochemistry. Number of mitochondria (numeric density: Nv), volume fraction occupied by mitochondria/microm3 of cytoplasm (volume density: Vv), and average mitochondrial volume (V) were calculated for both COX-positive and -negative organelles. The ratio (R) of the cytochemical precipitate area to the overall area of each mitochondrion was evaluated on COX-positive organelles to estimate the inner mitochondrial membrane fraction actively involved in cellular respiration. Following apoptotic stimulus, the whole mitochondrial population showed a significant increase of Nv and Vv, while V was significantly decreased. In COX-positive organelles higher values of Nv were found, V appeared significantly reduced, and Vv was unchanged. R was increased at a nonsignificant extent in apoptotic cells. COX-positive mitochondria accounted for 21% and 35% of the whole population in control and in apoptotic cells, respectively. These findings document that in the early stages of apoptosis the increased fraction of small mitochondria provides an adequate amount of ATP for progression of the programmed cell death and these more efficient organelles appear to represent a reactive response to the loss of metabolically impaired mitochondria. A better understanding of the mitochondrial role in neuronal apoptosis may suggest potential interventions to prevent the extensive nerve cell death typical of neurodegenerative diseases.

Age-related decline in metabolic competence of small and medium-sized synaptic mitochondria.

A computer-assisted morphometric investigation of cytochrome oxidase (COX) activity, selectively evidenced by preferential diaminobenzidine cytochemistry, has been carried out on synaptic mitochondria in the cerebellar cortex of adult and old rats. The ratio (R) of the area of the cytochemical precipitate (CPA) to the overall area of each mitochondrion (MA) was calculated. R refers to the fraction of the inner mitochondrial membrane actively involved in cellular respiration, thus its quantitative estimation constitutes a reliable index of the mitochondrial metabolic competence (MMC). In adult rats a significant negative correlation between MA and R values was found, while in old animals there was just a positive trend. Paired-quartile comparisons of R values showed a significant age-related decrease in small and medium-sized mitochondria, whereas the lowest and not significant age-related reduction was found in oversized organelles. A paired decrease in number and increase in size is reported to be a general trend for mitochondria during aging, but oversized organelles, according to their low R value, constitute a scanty, though functional, compensating reaction. Thus, the present findings support the argument that the currently reported age-related cellular metabolic decay appears to rely both on the decline in MMC of the small and medium-sized mitochondria, and on their specific reduction in number. This novel result is of biological relevance since it is largely the small and medium-sized mitochondria that are required for the provision of adequate amounts of ATP for actual cellular performance, while the significantly enlarged organelles are thought to represent an intermediate ultrastructural feature in mitochondrial genesis and/or remodelling.

Decay of mitochondrial metabolic competence in the aging cerebellum.

Cytochemically evidenced cytochrome oxidase activity was morphometrically measured in the cerebellar cortex of adult and old rats. The ratio (R) between the area of the precipitate due to the cytochemical reaction and the overall area of each mitochondrion was calculated. While in adult rats an inverse correlation between mitochondrial size and R values (r = -.905) was envisaged, in old animals increasing values of R were paired by increases in mitochondrial area (r =.561). Paired-quartile comparisons of the R values from adult and old animals documented a marked age-related impairment of the mitochondrial metabolic competence in small (I quartile: -31.6%) and medium-sized (II quartile: -26.4; III quartile: -16.4) mitochondria, while large organelles showed the lowest age-related decrease (IV quartile: -3.0%). The present findings support that a marked dysfunction of small and medium-sized mitochondria contributes to the significant decay of energy metabolism currently reported in physiological aging.

Cytochrome oxidase activity in hippocampal synaptic mitochondria during aging: a quantitative cytochemical investigation.

Synaptic mitochondria, cytochemically positive to cytochrome oxidase (COX) activity, were investigated by morphometric methods in the hippocampal dentate gyrus of adult and old rats. The number of mitochondria/microm(3) of tissue (Nv), the volume fraction occupied by mitochondria/microm(3) of tissue (Vv), the average mitochondrial volume (V), the longer mitochondrial diameter (F(max)), and the ratio R:mitochondrial area/overall area of the cytochemical precipitate due to COX activity were measured on COX-positive organelles. In old animals, Nv, Vv, V, and F(max) increased at a not significant extent; R was not significantly decreased. The complement (%) of longer organelles was higher in old animals. COX activity is currently considered an endogenous marker of neuronal oxidative metabolism; thus, although our findings refer to the discrete subpopulation of COX-positive organelles located at synaptic terminals, they support that changes of mitochondrial ultrastructure and metabolic competence may contribute to the age-related alterations of neuronal performances.

Chronic aluminum administration to old rats results in increased levels of brain metal ions and enlarged hippocampal mossy fibers.

The effect of chronic aluminium administration (2 g/L/6 months) was investigated in the central nervous system (CNS) of old rats. The content of Al(3+), Cu(2+), Zn(2+), and Mn(2+) was measured in prosencephalon + mesencephalon, pons-medulla, and cerebellum. The area occupied by the mossy fibers in the hippocampal CA3 zone was also measured. In Al-treated rats the contents of Al(3+), Cu(2+), Zn(2+), and Mn(2+) were significantly increased in prosencephalon + mesencephalon and pons-medulla, while no change was observed in the cerebellum except a Cu(2+) decrease. The area occupied by the mossy fibers in the CA3 field was significantly increased (+32%) in Al-treated rats. Taken together, the present findings document that the aging CNS is particularly susceptible to aluminum toxic effects that may be responsible for a consistent rise in the cell load of oxidative stress. This may contribute, as an aggravating factor, to the development of neurodegenerative events, as observed in Alzheimer disease.

Role of mitochondrial deterioration in physiological and pathological brain aging.

BACKGROUND: Mitochondria are widely reported to occupy a unique role in modulating cell viability, senescence and death. This is consistently supported by the multiple functions of these organelles. In addition to providing the energy for the myriad of cellular performances, mitochondria are involved in regulating thermogenesis, calcium buffering, integration of pro- and anti-apoptotic signals. OBJECTIVE: To stress the significant importance of subtle, continuous and permanent mitochondrial alterations as key events in physiological aging and as unfavourable determinants of age-related neurodegenerative diseases. RESULTS: Any dysfunction of these organelles may constitute a serious threat for cellular health status and survival, particularly of post-mitotic nerve and muscle cells. Mitochondrial deterioration may affect discrete features of the organelles (such as their structural dynamics, genetics and physiology) and lead to a progressive functional impairment. CONCLUSIONS: A variety of mitochondrial tasks, while hampering the possibility to recover the organelles' dysfunctions, offer different and reliable opportunities for therapeutic interventions.

GAP-43 mRNA detection by in situ hybridization, direct and indirect in situ RT-PCR in hippocampal and cerebellar tissue sections of adult rat brain.

The growth-associated protein GAP-43 is a presynaptic membrane phosphoprotein that is expressed at high levels during development and axonal growth. To evaluate the cellular distribution of GAP-43 mRNA in the hippocampus and cerebellum of adult rats we applied in situ hybridization (ISH) as well as direct and indirect in situ RT-PCR using biotin as a reporter molecule. ISH resulted in a positive signal in most cerebellar granular cells and in 30% of hippocampal CA3 neurons. Direct in situ RT-PCR yielded cells with strong signals in every region investigated, with elevated background levels most likely related to incorporation of labeled nucleotides into non-specific amplicons through internal priming and DNA repair activity. Indirect in situ RT-PCR turned out to be the best approach for detecting GAP-43 mRNA positive cells. Cerebellar cells exhibiting a positive signal for GAP-43 mRNA were of the granular cell type (98%). Hippocampal neurons with a positive reaction for GAP-43 mRNA included all the neuron groups analyzed, namely CA1 (99%) and CA3 pyramidal cells (94%) and dentate gyrus granule cells (92%). Dentate gyrus granule cells have not tested positive for GAP-43 mRNA detection by molecular morphology analysis. These data show that in normal rats GAP-43 mRNA is present in different cell populations of hippocampal formation, supporting the role of this protein in the ongoing processes of synaptic plasticity.

Inverse correlation between mitochondrial size and metabolic competence: a quantitative cytochemical study of cytochrome oxidase activity.

Mitochondria are topologically closed bilayered systems where the synthesis of adenosine triphosphate (ATP) from adenosine diphosphate (ADP) and inorganic phosphate occurs via oxidative phosphorylation. The ordered architecture (and its extension) of the mitochondria (i.e. inner membrane, outer membrane and cristae) constitutes a critical topographic arrangement for their energy-providing mechanisms. Thus, quantitative estimations of the ultrastructural features of organelles preferentially stained by means of function-related cytochemical reactions reliably report on their potential to supply adequate amounts of ATP. On the basis of this rationale, we carried out a computer-assisted cytochemical study of cytochrome oxidase (COX) activity on mitochondria of different size in the cerebellar cortex of adult rats. The total intra-mitochondrial area of the cytochemical precipitates (CPA)/mitochondrion, the area (MA) and the longer diameter (F(max)) of COX-positive organelles were measured. The ratio (R): CPA/MA was also calculated and referred to as the percentage of mitochondrial inner membrane area involved in COX activity. The regression analysis of R vs MA showed a significant inverse correlation (r=-0.905). The fourfold increase in MA from quartiles I to IV was matched by increases in F(max) and CPA, respectively, but it was also related to a 25% decrease in R. By matching quantitative cytochemical estimations of COX activity within mitochondria with the morphometric assessment of their ultrastructural features, the present study correlates size to the metabolic competence of COX-positive organelles. Quantitative cytochemistry of COX activity is currently regarded as a reliable marker of cellular metabolism; thus our findings support the hypothesis that enlargements in size are inversely correlated with the mitochondrial metabolic competence.

Neuronal death versus synaptic pathology in Alzheimer's disease.

Neuron and synapse numeric densities as well as the average size and surface density of the synaptic junctional areas were measured in the hippocampus and cerebellum of adult, old, and demented (Alzheimer's disease) patients. Our findings support the notion that synaptic loss represents per se a prominent and early damage affecting zones of the central nervous system reported to show a different vulnerability to age- and pathology-related changes.