Elena Radi

Mitochondria, oxidative stress and neurodegeneration

Mitochondria are involved in ATP supply to cells through oxidative phosphorylation (OXPHOS), synthesis of key molecules and response to oxidative stress, as well as in apoptosis. They contain many redox enzymes and naturally occurring inefficiencies of oxidative phosphorylation generate reactive oxygen species (ROS). CNS functions depend heavily on efficient mitochondrial function, since brain tissue has a high energy demand. Mutations in mitochondrial DNA (mtDNA), generation and presence of ROS and environmental factors may contribute to energy failure and lead to neurodegenerative diseases. Many rare metabolic disorders have been associated with mitochondrial dysfunction. More than 300 pathogenic mtDNA mutations involve proteins that regulate OXPHOS and mitochondrial structural integrity, and have also been described in neurodegenerative diseases with autosomal inheritance. Mitochondria may have an important role in ageing-related neurodegenerative disorders like Parkinsons disease (PD), Alzheimers disease (AD), Huntingtons disease (HD) and amyotrophic lateral sclerosis (ALS). In primary mitochondrial and neurodegenerative disorders, there is strong evidence that mitochondrial dysfunction occurs early and has a primary role in pathogenesis. In the present review, we discuss several mitochondrial diseases as models of neurodegeneration.

Apoptosis and Oxidative Stress in Neurodegenerative Diseases

Neurodegenerative disorders affect almost 30 million individuals leading to disability and death. These disorders are characterized by pathological changes in disease-specific areas of the brain and degeneration of distinct neuron subsets. Despite the differences in clinical manifestations and neuronal vulnerability, the pathological processes appear similar, suggesting common neurodegenerative pathways. Apoptosis seems to play a key role in the progression of several neurologic disorders like Alzheimers disease, Parkinsons disease, Huntingtons disease, and amyotrophic lateral sclerosis as demonstrated by studies on animal models and cell lines. On the other hand, research on human brains reported contradictory results. However, many dying neurons have been detected in brains of patients with neurodegenerative diseases, and these conditions are often associated with significant cell loss accompanied by typical morphological features of apoptosis such as chromatin condensation, DNA fragmentation, and activation of cysteine-proteases, caspases. Cell death and neurodegenerative conditions have been linked to oxidative stress and imbalance between generation of free radicals and antioxidant defenses. Multiple sclerosis, stroke, and neurodegenerative diseases have been associated with reactive oxygen species and nitric oxide. Here we present an overview of the involvement of neuronal apoptosis and oxidative stress in the most important neurodegenerative diseases, mainly focusing the attention on several genetic disorders, discussing the interaction between primary genetic abnormalities and the apoptotic pathways.

Cerebrospinal fluid tau, Aß, and phosphorylated tau protein for the diagnosis of Alzheimer's disease

The diagnosis of AD is still largely based on exclusion criteria of secondary causes and other forms of dementia with similar clinical pictures, than the diagnostic accuracy of AD is low. Improved methods of early diagnosis are needed, particularly because drugs treatment is more effective in the early stages of the disease. Recent research focused the attention to biochemical diagnostic markers (biomarkers) and according to the proposal of a consensus group on biomarkers, three candidate CSF markers reflecting the pathological AD processes, have recently been identified: total tau protein (t‐tau), amyloid ß(1–42) protein (Aß42), and tau protein phosphorylated at AD‐specific epitopes (p‐tau). Several articles report reduced CSF levels of Aß42 and increased CSF levels of t‐tau and p‐tau in AD; the sensitivity and specificity of these data are able for discrimination of AD patients from controls. However, the specificity for other dementias is low. According to the literature analysis reported in the present review, we can conclude that the combination of the CSF markers and their ratios may significantly increase the specificity and the accuracy of AD diagnosis.

Psychosine-induced apoptosis and cytokine activation in immune peripheral cells of Krabbe patients†

Globoid cell leukodystrophy or Krabbe disease (KD), is a hereditary disorder caused by galactosylceramidase deficiency. Progressive accumulation of psychosine is considered to be the critical pathogenetic mechanism of cell death in the Krabbe brain. Psychosine mechanism of action has not been fully elucidated. It seems to induce apoptosis in oligodendrocytes through a mitochondrial pathway and to up‐regulate inflammatory cytokines production resulting in oligodendrocyte loss. Our aim was to evaluate the role of psychosine in apoptotic cell death and inflammatory response in a group of patients affected by KD using peripheral blood lymphocytes (PBLs) and peripheral blood mononuclear cells (PBMCs) as a cellular model. PBLs from KP and healthy controls were exposed to 20 µM psychosine and analysed by flow cytometry, agarose gel electrophoresis and fluorescence microscopy. Our results showed that psychosine induces apoptosis in PBLs through a mitochondrial pathway, but the apoptotic response was quite low especially KP. The role of psychosine in the up‐regulation of cytokines (TNFalpha, IL8 and MCP1) has been evaluated by ELISA in PBMCs from KP and controls after stimulation with LPS and phytohemagglutinin. Both in basal condition and after LPS stimulation, cells from KP showed a significant increase in TNF‐α production, reduced MCP1 levels and no modification in IL8. These results indicate that lymphomonocytes from KP had a basal proinflammatory pattern that was amplified by psychosine. In conclusion, the reduced apoptotic response and the atypical cytokine production observed in our experiments, suggest an involvement of inflammatory pattern in immune peripheral cells of KP. J. Cell. Physiol. 212:737–743, 2007.

Human fibroblasts undergo oxidative stress‐induced apoptosis without internucleosomal DNA fragmentation

In order to evaluate the reliability of fibroblasts as a cell model for studying apoptosis, we tested the response of normal human fibroblasts to the oxidative stress inducers H2O2 and 2‐deoxy‐D‐ribose (dRib). Our results showed that fibroblasts treated with dRib and H2O2 are induced to undergo apoptosis as demonstrated by reduction in total cell number, chromatin condensation, phosphatidylserine (PS) exposure, activation of caspase‐3 and 7, changes in mitochondrial membrane potential and increase in the number of terminal deoxynucleotidyl transferase‐mediated dUTP nick end‐labeling (TUNEL)‐positive nuclei. However we only found a slight increase in the percentage of cells in the sub‐G1 region evaluated by flow cytometry, and we did not observe DNA fragmentation by agarose gel electrophoresis. Early in apoptosis, DNA cleavage generates high molecular weight (HMW) fragments which can be detected by TUNEL assay; successively followed by a pronounced DNA brake down into low molecular weight (LMW) fragments, detected as a “DNA ladder” by conventional agarose gel electrophoresis and as an hypodiploid peak by propidium iodide (PI) flow cytometry assay. Our results thus suggest that only HMW fragmentation occurs in fibroblasts exposed to dRib or H2O2 and the lack of internucleosomal DNA fragmentation may depend on the peculiar characteristics of human fibroblasts themselves, irrespective of the apoptotic stimulus used. The existence of distinct events leading to cell death in different cell types makes it necessary to use a combination of strategies and techniques to evaluate the occurrence of apoptosis. J. Cell. Physiol. 208: 289–297, 2006.

Apoptosis in CADASIL: An in vitro study of lymphocytes and fibroblasts from a cohort of Italian patients

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a hereditary disease affecting vascular smooth muscle cells of nearly all tissues. Clinical manifestations mainly concern the central nervous system with repeated TIA/stroke, migraine, psychiatric disturbances, and cognitive decline. Minor findings have been reported in muscle, nerve, and skin. CADASIL is due to NOTCH3 gene mutations. This gene has been identified as an up‐regulator of c‐FLIP, an inhibitor of Fas‐ligand‐induced apoptosis. The aim of this study was to assess the involvement of oxidative stress‐induced apoptosis in cells from 16 Italian CADASIL patients. Peripheral blood lymphocytes (PBLs) and fibroblasts from CADASIL patients were exposed to 2‐deoxy‐D‐ribose (dRib), which induces apoptosis by oxidative stress. Apoptosis was analyzed by flow cytometry, agarose gel electrophoresis and fluorescence microscopy for caspase‐3 activation, phosphatidylserine exposure and mitochondrial membrane depolarization. PBLs and fibroblasts from CADASIL patients showed a significantly higher response to dRib‐induced apoptosis than those of controls. PBLs from CADASIL patients also showed a significantly higher percentage of apoptotic cells than PBLs from controls, even when cultured without dRib. The greater susceptibility of PBLs and fibroblasts from CADASIL patients to dRib‐induced apoptosis suggests that NOTCH3 mutations are an important apoptotic trigger. Since PBLs from patients showed higher levels of apoptosis even in the absence of an apoptotic stimulus, cells from CADASIL patients appear to be physiologically prone to apoptotic cell death. J. Cell. Physiol. 219: 494–502, 2009.

CSF Biomarkers Profile in CADASIL-A Model of Pure Vascular Dementia: Usefulness in Differential Diagnosis in the Dementia Disorder.

Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is considered a model of pure vascular dementia (VD) because it occurs in young adults unlikely to have concomitant age and Alzheimers Disease-(AD-) related pathology. CSF levels of β-amyloid 1-42 (Aβ42), total tau protein (t-tau), and phosphorylated tau-protein (p-tau), well accepted biomarkers of AD, were evaluated in 10 CADASIL patients, 22 AD patients, and 17 healthy age-matched subjects. Innotest β-amyloid 1-42, Innotest hTAU-Ag, and Innotest Phospho-tau 181p sandwich enzyme-linked immunoassay were used to determine CSF biomarkers levels. A case-control statistical analysis was carried out. CSF Aβ42 levels were significantly lower in CADASIL patients and considerable overlap with AD whereas t-tau and p-tau levels were normal and significantly different with respect to AD. A significant altered CSF biomarkers profile in a pure VD supports the use of CSF Aβ42, t-tau, and p-tau levels in the differential diagnosis of VD and AD.

CSF levels of β-amyloid 1-42, tau and phosphorylated tau protein in CADASIL

Background and purpose:  Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) can be considered a useful model of pure subcortical vascular dementia (SVD) because it occurs in young adults, unlikely to have concomitant age‐ and Alzheimer’s disease (AD)‐related pathology. In patients with CADASIL we evaluated the cerebrospinal fluid (CSF) levels of β‐amyloid 1‐42 (Aβ42), total tau protein (t‐tau) and phosphorylated tau protein (p‐tau), which are well‐accepted biomarkers of AD.

Cerebrolysin administration reduces oxidative stress-induced apoptosis in limphocytes from healthy individuals

Cerebrolysin is the only drug available for clinical use containing active fragments of some important neurotrophic factors obtained from purified porcine brain proteins, which has long been used for the treatment of dementia and stroke sequels. Cerebrolysin has growth factor‐like activities and promotes neuronal survival and sprouting, however, its molecular mechanism still needs to be determined. It has been shown that Cerebrolysin may interact with proteolytic pathways linked to apoptosis. Administration of Cerebrolysin significantly reduces the number of apoptotic neurons after glutamate exposure. Furthermore, it has been reported that Cerebrolysin inhibits free radicals formation and lipid peroxidation. In vitro we evaluated the protective effects of Cerebrolysin towards spontaneous and induced apoptotic death in cells from healthy individuals. Peripheral blood lymphocytes (PBLs) from 10 individuals were used as cell model; 2‐deoxy‐D‐ribose (dRib), a highly reducing sugar, was used as paradigm pro‐apoptotic stimulus. Apoptosis was analysed using flow cytometry and fluorescence microscopy. Our results showed that Cerebrolysin significantly reduced the number of apoptotic PBLs after dRib treatment, although it had no significative effects on cells cultured in standard conditions. Our work showed a protective effect of Cerebrolysin on oxidative stress‐induced apoptosis and suggested that PBLs can be used as an easy obtainable and handy cell model to verify Cerebrolysin effects in neurodegenerative pathologies.

Altered apoptosis regulation in Kufor–Rakeb syndrome patients with mutations in the ATP13A2 gene

ATP13A2 gene encodes for a protein of the group 5 P‐type ATPase family. ATP13A2 mutations are responsible for Kufor–Rakeb syndrome (KRS), a rare autosomal recessive juvenile parkinsonism characterized by the subacute onset of extrapyramidal, pyramidal and cognitive dysfunction with secondary nonresponsiveness to levodopa. FBXO7 protein is an F‐box‐containing protein. Recessive FBXO7 mutations are responsible for PARK15, a rare juvenile parkinsonism characterized by progressive neurodegeneration with extrapyramidal and pyramidal system involvement. Our aim was to evaluate apoptosis in cells from two KRS siblings carrying a homozygous ATP13A2 mutation and a heterozygous FBXO7 mutation. We also analysed apoptosis in the patients’ healthy parents. Peripheral blood lymphocytes from the KRS patients and parents were exposed to 2‐deoxy‐D‐ribose; apoptosis was analysed by flow cytometry and fluorescence microscopy. Apoptosis was much higher in lymphocytes from the KRS patients and parents than in controls, both in standard conditions and after induction with a pro‐apoptotic stimulus. The lack of correlation between increased apoptosis and the presence of the mutated FBXO7 gene rules out the involvement of FBXO7 in apoptosis regulation. The altered apoptotic pattern of subjects with mutated ATP13A2 suggests a correlation between apoptosis alteration and the mutated ATP13A2 protein. We hypothesize that ATP13A2 mutations may compromise protein function, disrupting cell cation balance and rendering cells prone to apoptosis. However, the deregulation of apoptosis in KRS patients displaying different disease severity suggested that the altered apoptotic pathway probably does not have a pathogenetic role in KRS by itself.