Moreno Solazzi

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 A2 (PLA2) 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.

Distribution of MAP2 in Hippocampus and Cerebellum of Young and Old Rats by Quantitative Immunohistochemistry

The microtubule-associated protein MAP2 is a cytoskeletal protein that plays a regulatory role in neuronal plasticity and in maintaining the morphology of differentiated neurons. MAP2 distribution was assessed in hippocampus and cerebellum of young and old rats by quantitative immunohistochemistry. In old vs young rats, densitometric analysis showed a significant decrease of MAP2 immunoreactivity in the hippocampus CA1 field (-93%), whereas no difference was found in cerebellar MAP2 distribution. These preliminary data suggest that in areas of the brain involved in memory acquisition and consolidation, MAP2-dependent neuroplasticity and structural integrity are significantly decreased in aging. (J Histochem Cytochem 49:1065–1066, 2001)

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.

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 (Fmax) 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 Fmax 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.

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.

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.

Quantitative cytochemical mapping of mitochondrial enzymes in rat cerebella

Mitochondrial metabolic competence, defined as the organelles capacity to provide adequate amounts of ATP in due time, appears to constitute an important determinant in several biological processes and pathological conditions. Thus, the assessment of the metabolic efficiency of the mitochondrial population in a given tissue area or cellular compartment may provide clues to identifying alterations of the cellular bioenergetic machinery, which may constitute a predisposing condition leading to impaired organ and system functions. In the cerebellar cortex of adult rats, the activities of the enzymes cytochrome oxidase (COX) and succinic dehydrogenase (SDH) were, respectively, evidenced by means of the diaminobenzidine and copper ferrocyanide preferential cytochemical techniques. At the electron microscope, the activities of these two key molecules of the respiratory chain were clearly visualised as dark precipitates at the inner mitochondrial membrane sites where COX and SDH are located. By means of the disector method, unbiased mitochondrial samplings were carried out to measure: the number of mitochondria/microm(3) of tissue (numeric density: Nv); the mitochondrial volume fraction/microm(3) of tissue (volume density: Vv) and the average mitochondrial volume (V) both on COX- and SDH-positive organelles in the cerebellar glomeruli and Purkinje cells, respectively. The ratio R (total area of the precipitates due either to COX or SDH activity within the single mitochondrion/area of the same organelle) was also evaluated to get information on the enzyme activity related to mitochondrial size.The documented accumulation of mutant mitochondrial DNA particularly in postmitotic cells results in a marked heteroplasmy (mixtures of normal and mutated genomes) at mitochondrial and cellular levels, thus the cellular potential for energy production is demanded to a mosaic of organelles with different functional capabilities. Assessment of the mitochondrial mosaic outline by means of quantitative cytochemistry of key enzymes of the respiratory chain, such as COX and SDH, may allow for the morphofunctional metabolic mapping of mitochondrial efficiency in discrete cellular or tissue compartments.

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 Alzheimers 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-β (Aβ) 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.

Neuronal Death versus Synaptic Pathology in Alzheimer's Disease

Abstract: 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 (Alzheimers 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.

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.