Patrizia Formichi

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.

Cell response to oxidative stress induced apoptosis in patients with Leber’s hereditary optic neuropathy

Objectives: Leber’s hereditary optic neuropathy (LHON) is a maternally inherited disease in which acute or subacute bilateral visual loss occurs preferentially in young men. Over 95% of LHON cases are associated with one of three mitochondrial DNA (mtDNA) point mutations, but only 50% of men and 10% of women who harbour a pathogenetic mtDNA mutation develop optic neuropathy. This incomplete penetrance and preference for men suggests that additional genetic (nuclear or mitochondrial) and/or environmental factors must modulate phenotype expression in LHON. A role for reactive oxygen species (ROS) in mitochondrial diseases, secondary to mtDNA mutations, or as a result of the direct effect of ROS cytotoxicity, has been implicated in many mitochondrial disorders, including LHON. The purpose of this study was to investigate the role of oxidative stress induced apoptosis in LHON. Methods: The 2-deoxy-D-ribose induced apoptotic response of peripheral blood lymphocytes from six patients with LHON and six healthy subjects was investigated using light microscopy, flow cytometry, agarose gel electrophoresis, and the measurement of mitochondrial membrane potential. Results: Cells of patients with LHON had a higher rate of apoptosis than those of controls and there was evidence of mitochondrial involvement in the activation of the apoptotic cascade. Conclusions: These differences in oxidative stress induced apoptosis are in line with the hypothesis that redox homeostasis could play a role in the expression of genetic mutations in different individuals and could represent a potential target in the development of new therapeutic strategies.

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.

Apoptotic response and cell cycle transition in ataxia telangiectasia cells exposed to oxidative stress.

The recently identified ATM gene plays a role in a signal transduction network activating multiple cellular functions in response to DNA damage. An attractive hypothesis is that the ATM protein is involved in a specialized antioxidant system responsible for detoxifying reactive oxygen intermediate and that the absence or dysfunction of this protein in AT cells would render them less capable of dealing with oxidative stress. In order to investigate the role of the ATM gene in cell cycle control and programmed cell death, Lymphoblastoid cell lines derived from four Ataxia-Telangiectasia (AT) patients and six controls have been analyzed. All cell lines were incubated with 2-deoxy-D-ribose (dRib), a reducing sugar that induces apoptosis through oxidative stress. The result showed an impaired response to dRib-induced apoptosis in AT cells, as well as a defect of cellular cycle arrest in G1/S phase and a normal expression of p53 protein. This indicate that the kinase activity of ATM gene product plays a very important role in the cellular response to oxidative stress. In conclusion the altered response of AT cells to oxidative stress and particularly their resistance to apoptotic cell death, could explain the high predisposition of these cells to progress toward malignant transformation.

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.

Evidence of diffuse brain pathology and unspecific genetic characterization in a patient with an atypical form of adult-onset Krabbe disease

Sirs: Krabbe disease (KD) is a rare autosomal recessive disorder due to a diminished activity of the lysosomal enzyme galactocerebrosidase (GALC) [7, 13]. The demonstration of a severe deficiency in GALC activity in leukocytes or fibroblasts is the basis for diagnosis. The human GALC gene (mapped to chromosome 14q24.3– q32.1, see [14] for details) has been cloned, and a number of KD-causing mutations have been identified. Recent studies have reported an unusual, slowly progressive adult form of KD characterized by spastic paraparesis and magnetic resonance imaging (MRI) findings of bilateral corticospinal tract demyelination [11, 12]. It has been suggested that the new homozygous point mutation T1835C (L618S) found in the GALC gene of one of these patients [11] is characteristic of this form of late-onset KD. We describe a 44-year-old woman who was hospitalized because of slowly progressive gait disturbance. There was no family history of neurological disease. Her birth and delivery were uncomplicated, and development milestones were normal. She did not have neurological symptoms until the age of 42 years, when she began to complain of an unsteadiness of gait. Neurological examination showed mild spastic paraparesis with increased muscle tone in the legs. All deep-tendon reflexes were symmetrically brisk, with bilateral Babinski sign. Sense of joint position and sensation to light touch and pinprick were normal, while vibration sense was mildly decreased in the legs. Mental functions were normal. Blood laboratory examinations showed an iron deficiency anemia. Urine tests were normal. Electromyography and nerve conduction studies showed evidence of demyelinating sensorimotor neuropathy. A sural nerve biopsy specimen revealed mild signs of demyelination, with the presence of inclusions within Schwann’s cells on electron microscopy. The activity of a number of lysosomal enzymes was screened in the patient’s cultured skin fibroblasts [15], and only the GALC activity was decreased (0.26 nmol h–1 mg–1 protein, <10% of the normal mean). DNA was isolated from cultured cells as previously described [10]. The patient was found to be heterozygous for two previously reported mutations: G > A at cDNA position 809 (G270D) and 1026del10. The first mutation, G > A at cDNA position 809 (G270D), has been identified previously in other patients with adult-onset KD [3, 6, 14]. The second mutation, previously identified in another Italian patient with infantile KD [14], consists of a 10 basepair deletion starting in exon 10 at nucleotide 1026. This results in a frame shift and premature stop codon and most likely would not produce any active enzyme since there would be no production of the 30-kDa subunit necessary for activity. Combined brain proton MRI and magnetic resonance spectroscopic imaging (MRSI) was performed using a Philips Gyroscan NT operating at 1.5 T. Multislice spin-echo images were obtained in coronal and transverse planes perpendicular and parallel to the anterior commissure (AC) and posterior commissure (PC) line, LETTER TO THE EDITORS J Neurol (2000) 247 :226–228