Giovanni Stefanoni

Reduced expression of the chaperone-mediated autophagy carrier hsc70 protein in lymphomonocytes of patients with Parkinson's disease

Chaperone-mediated autophagy (CMA) impairment is recognized to play a pathogenetic role in Parkinsons disease (PD). A reduced expression of lysosomal-associated membrane protein (lamp) 2A and heat shock cognate (hsc) 70 protein, the two key regulators of CMA, has been reported in brains of PD patients. To verify the existence of a possible systemic CMA dysfunction in PD, in this study the expression of hsc70 and lamp2A was assessed in peripheral blood mononuclear cells (PBMC) of patients with sporadic PD and compared to healthy subjects. The expression of myocyte enhancer factor 2D (MEF2D), a transcriptional factor implicated in neuronal survival and specific substrate of CMA, was also evaluated. Protein and gene expression was assessed by Western blot and real-time PCR, respectively, in PBMC obtained from 53 sporadic PD patients and 53 healthy subjects. A significant reduction of hsc70 levels was observed in PBMC of PD patients, both under basal conditions and after autophagy induction obtained with serum deprivation. No difference emerged in lamp2A and MEF2D expression between patients and controls. No influence of the clinical characteristics of patients emerged on hsc70, lamp2A and MEF2D expression. These results, despite being not suggestive of the existence of a CMA impairment in PBMC of PD patients, identify a systemic hsc70 reduction in PD patients. Further studies on specific mechanisms and biological significance of such alteration are needed to corroborate this finding that could lead to the identification of a new trait biomarker for PD.

Rotenone upregulates alpha-synuclein and myocyte enhancer factor 2D independently from lysosomal degradation inhibition.

Dysfunctions of chaperone-mediated autophagy (CMA), the main catabolic pathway for alpha-synuclein, have been linked to the pathogenesis of Parkinsons disease (PD). Since till now there is limited information on how PD-related toxins may affect CMA, in this study we explored the effect of mitochondrial complex I inhibitor rotenone on CMA substrates, alpha-synuclein and MEF2D, and effectors, lamp2A and hsc70, in a human dopaminergic neuroblastoma SH-SY5Y cell line. Rotenone induced an upregulation of alpha-synuclein and MEF2D protein levels through the stimulation of their de novo synthesis rather than through a reduction of their CMA-mediated degradation. Moreover, increased MEF2D transcription resulted in higher nuclear protein levels that exert a protective role against mitochondrial dysfunction and oxidative stress. These results were compared with those obtained after lysosome inhibition with ammonium chloride. As expected, this toxin induced the cytosolic accumulation of both alpha-synuclein and MEF2D proteins, as the result of the inhibition of their lysosome-mediated degradation, while, differently from rotenone, ammonium chloride decreased MEF2D nuclear levels through the downregulation of its transcription, thus reducing its protective function. These results highlight that rotenone affects alpha-synuclein and MEF2D protein levels through a mechanism independent from lysosomal degradation inhibition.

Rotenone down-regulates HSPA8/hsc70 chaperone protein in vitro: a new possible toxic mechanism contributing to Parkinson's disease

HSPA8/hsc70 (70-kDa heat shock cognate) chaperone protein exerts multiple protective roles. Beside its ability to confer to the cells a generic resistance against several metabolic stresses, it is also involved in at least two critical processes whose activity is essential in preventing Parkinsons disease (PD) pathology. Actually, hsc70 protein acts as the main carrier of chaperone-mediated autophagy (CMA), a selective catabolic pathway for alpha-synuclein, the main pathogenic protein that accumulates in degenerating dopaminergic neurons in PD. Furthermore, hsc70 efficiently fragments alpha-synuclein fibrils in vitro and promotes depolymerization into non-toxic alpha-synuclein monomers. Considering that the mitochondrial complex I inhibitor rotenone, used to generate PD animal models, induces alpha-synuclein aggregation, this study was designed in order to verify whether rotenone exposure leads to hsc70 alteration possibly contributing to alpha-synuclein aggregation. To this aim, human SH-SY5Y neuroblastoma cells were treated with rotenone and hsc70 mRNA and protein expression were assessed; the effect of rotenone on hsc70 was compared with that exerted by hydrogen peroxide, a generic oxidative stress donor with no inhibitory activity on mitochondrial complex I. Furthermore, the effect of rotenone on hsc70 was verified in primary mouse cortical neurons. The possible contribution of macroautophagy to rotenone-induced hsc70 modulation was explored and the influence of hsc70 gene silencing on neurotoxicity was assessed. We demonstrated that rotenone, but not hydrogen peroxide, induced a significant reduction of hsc70 mRNA and protein expression. We also observed that the toxic effect of rotenone on alpha-synuclein levels was amplified when macroautophagy was inhibited, although rotenone-induced hsc70 reduction was independent from macroautophagy. Finally, we demonstrated that hsc70 gene silencing up-regulated alpha-synuclein mRNA and protein levels without affecting cell viability and without altering rotenone- and hydrogen peroxide-induced cytotoxicity. These findings demonstrate the existence of a novel mechanism of rotenone toxicity mediated by hsc70 and indicate that dysfunction of both CMA and macroautophagy can synergistically exacerbate alpha-synuclein toxicity, suggesting that hsc70 up-regulation may represent a valuable therapeutic strategy for PD.

A panel of macroautophagy markers in lymphomonocytes of patients with amyotrophic lateral sclerosis

Abstract A potential role for macroautophagy dysfunction in the pathogenesis of amyotrophic lateral sclerosis (ALS) was hypothesized after the demonstration that selected markers are up-regulated in post mortem samples obtained from both patients and animal models of disease. We hypothesized that a putative dysfunction of this catabolic pathway could be operative also in peripheral blood mononuclear cells (PBMC) obtained from ALS patients, since these cells represent an accessible model for studying molecular pathogenesis events in neuropsychiatric disorders. Beclin-1 and LC3II immunoreactivity were assessed in PBMC from 15 ALS patients and 15 controls by Western blot analysis. The expression of Atg12 mRNA was also assessed by real-time PCR. No significant difference was observed for all these parameters between patients and controls, although PBMC displayed a clear macroautophagy induction following exposure to rapamycin and lithium. Finally, we excluded a putative interference of riluzole demonstrating that LC3II immunoreactivity did not change in riluzole-treated SH-SY5Y neuroblastoma cells. In conclusion, the results of our pilot study do not support the idea of a systemic macroautophagic dysfunction in ALS, although they confirm that PBMC are a suitable peripheral marker for monitoring the effects of drugs interfering with this catabolic pathway.

Alpha-Synuclein, Oxidative Stress and Autophagy Failure: Dangerous Liaisons in Dopaminergic Neurodegeneration

The molecular mechanisms of neurodegeneration in Parkinson’s disease and the cause of the selective dopaminergic neuronal loss are mostly unknown. Many pathogenetic factors have been found to play a role but the relationships among these factors, together with the reasons of the high vulnerability of dopaminergic neurons to them, have not been completely defined. Only a small fraction of Parkinson’s disease cases have a defined etiology: this fraction include the monogenic hereditary variants of the disease and the sporadic cases determined by prolonged exposition to toxic agents inhibiting mitochondrial complex I, such as 1,1’-dimethyl-4,4’-5 bipyridinium (paraquat), rotenone and 1-methyl-4phenyl-1,2,3,6-tetrahydropyridine (MPTP). Parkinson’s disease-related toxins and pathogenetic mutations have been indispensable to create cell and animal models with the aim to clarify the molecular physiopathology of the disease. Little is known about the primitive causes of idiopathic Parkinson’s disease, that probably represents a multi-factorial disease influenced by various genetic and environmental factors, all characterized by high incidence in general population. The different risk factors together would contribute to initiate the complex pathogenetic sequence of events leading to the death of dopaminergic neurons. Recently, Parkinson’s disease has been placed in the large category of neurodegenerative diseases caused by protein misfolding. In particular, alpha-synuclein has been proposed as the central and most specific factor implied in the pathogenesis of this syndrome, which, as a consequence, has been classified among synucleinopathies, together with dementia with Lewy bodies and multiple system atrophy, other neurodegenerative diseases having alphasynuclein pathology as a major feature. Aim of this chapter is to provide an organic revision of evidences for the involvement of alpha-synuclein in the pathogenesis of Parkinson’s disease. We will define the mechanisms responsible for the toxic gain of function of ┙-synuclein and the processes triggered by aberrant alpha-synuclein and mediating its neurotoxic effect. Particular attention will be paid to establish the links that correlate the deleterious action of alpha-synuclein with oxidative stress and with the efficiency of the processes involved in the clearance of aberrant

Back to the ring: knocking-out headache.

On October 2010, a 32-year-old Albanian man, living in Italy since 2001, came to the emergency department of our hospital reporting a several months history of persistent sub-continuous occipital headache unresponsive to pharmacological therapy. He worked as bricklayer and he used to go back to Albany every 4 months; the last visit to his country had occurred 1 month before. The patient was apparently in good health, with no remarkable history for relevant diseases. He denied smoking, alcohol or drug use. His mother and his two brothers were apparently in good health too; his father had died of gastric cancer. On physical examination the patient was afebrile, without lymphadenopathy, fully conscious, orientated and showed no overt neurological abnormalities. Haematology and basic blood chemistry tests gave no pathological results; he was negative for HIV infection. A contrast-enhanced CT scan of the brain revealed a hyperdense parafalcial posterior single lesion with ring enhancement, initially interpreted as a possible meningioma. The patient was admitted to the Neurology ward and a brain MRI showed three small rounded lesions in the right hemisphere, all with a vivid gadolinium peripheral enhancement (the largest lesion within the superior frontal gyrus is shown in Fig. 1a).

Jasmine smell hides an unwelcome surprise: a case of paraneoplastic limbic encephalitis presenting with olfactory epileptic auras

Mesial temporal lobe epilepsy (MTLE) usually appears in adolescence as a consequence of idiopathic hippocampal sclerosis. The typical manifestations are complex partial seizures with automatisms and loss of awareness, often preceded by simple seizures, named auras [1, 2]; in some cases the latter are the unique symptom of presentation. The most frequently reported auras are rising epigastric sensations and emotional phenomena, such as fear. Olfactory or gustatory dysperceptions are less frequent. In a clinical series, only 5% of patients with MTLE had olfactory auras [3], which are probably generated by epileptic activity in the amygdala, although the olfactory bulb may be another possible epileptogenic zone [4]. Piloerection is reported even less frequently and represents a manifestation of autonomic seizures [5]. Here, we describe a patient with stereotyped episodes of multimodal auras, including olfactory, emotional and autonomic symptoms, without subsequent loss of consciousness. A 67 year-old man was admitted for a 3 month-history of brief olfactory dysperceptions. He described episodes occurring 5–10 times a day and lasting about 90 s, with a stereotyped presentation: an initial jasmine perfume, followed by a nasty smell of pepper, and, finally, by a worrying sensation of fear accompanied by diffuse piloerection. Neurologic examination was negative for focal deficits during and between episodes. No deficits in short and long-term memory, speech, executive functions or other cognitive functions were found. Patient did not show any abnormality in behavior and mood. Despite a negative EEG, episodes were interpreted as partial temporal seizures so that carbamazepine was started with complete resolution of the episodes. Brain MRI demonstrated hyperintensity of the left mesial hippocampus and amygdala on T2 and FLAIR sequences, with minimal cortical swelling and contrast-enhancement (Fig. 1a, c). DWI sequences did not show signs of cyotoxic edema. A diagnosis of probable low-grade glioma was hypothesized, and the patient was going to be referred to the neurosurgeon for biopsy. Moreover, given his medical history of heavy smoking (120 pack-years) and recent significant weight loss (15 kg in the last 3 months), other investigations were made. A lumbar puncture revealed elevated CSF proteins (96 mg/dL). A panel of blood exams showed mild increase of neuron specific enolase. Exams were completed by plasma testing for onconeural antibodies including antiHu, Ri, Yo, amfifisin, CV2 and PNMA2. Test gave positive result for anti-Hu, so a paraneoplastic limbic encephalitis was suspected. Computed tomography demonstrated a mass lesion in the right lung: the subsequent biopsy led to the demonstration of a small cell lung cancer. Metastasis were excluded and patient underwent four cycles of chemotherapy followed by radiotherapy with important reduction of tumor size and metabolic activity. About 6 months later brain MRI was repeated: the brain lesion was still visible but cortical swelling and contrast-enhancement had disappeared (Fig. 1b, d). This evolution led to the definitive exclusion of glioma, supporting the diagnosis of limbic encephalitis. MTLE in adults has two most frequent causes: tumors and encephalitis involving mesial temporal lobe. Limbic & Giovanni Stefanoni giovanni_stefanoni@alice.it

Exploring the Role of Autophagy in the Pathogenesis of Rotenone-induced Toxicity

This chapter is intended to provide an organic revision of evidence for the involvement of autophagy in the pathogenesis of rotenone-induced toxicity. The mechanisms underlying rotenone neurotoxicity are elucidated through a description of findings obtained in both in vivo and in vitro experimental models. Furthermore, this chapter describes the etiological role of rotenone and other pesticide exposure in the pathogenesis of Parkinson’s disease, as demonstrated by epidemiological studies performed in the last decades starting from results obtained in animal and cellular experimental models. The specific focus of the present chapter is to explore the effect of rotenone on autophagic pathway, whose dysfunctions are already recognized to play an important pathogenetic role in Parkinson’s disease. Specifically, a comprehensive revision of the current available literature on the rotenone-induced dysfunctions of the two main types of autophagy, macroautophagy and chaperone-mediated autophagy, is provided in order to clarify the protective rather than detrimental contribute exerted by autophagy alterations in the cell death induced by rotenone.

Brain Targets: Can You Believe Your Own Eyes?

The unquestionable advantages provided by modern neuroimaging techniques have recently led some to question the duty of the neurologist, traditionally struggling first and foremost to establish the semeiotic localization of brain lesions and only then to interpret them. The present brief report of six clinical patients who came recently to our attention aims to emphasize that the interpretation of neuroimaging results always requires integration with anamnestic, clinical and laboratory data, together with knowledge of nosography and the literature. The solutions of the reported cases always originated from close interaction between the neurologist and the neuroradiologist, based on the initial diagnostic uncertainty linked to the finding of isolated or multiple brain target or ring lesions, too often considered paradigmatic examples of the pathognomonic role of neuroimaging.