Marco Spinazzi

Assessment of mitochondrial respiratory chain enzymatic activities on tissues and cultured cells

The assessment of mitochondrial respiratory chain (RC) enzymatic activities is essential for investigating mitochondrial function in several situations, including mitochondrial disorders, diabetes, cancer, aging and neurodegeneration, as well as for many toxicological assays. Muscle is the most commonly analyzed tissue because of its high metabolic rates and accessibility, although other tissues and cultured cell lines can be used. We describe a step-by-step protocol for a simple and reliable assessment of the RC enzymatic function (complexes I–IV) for minute quantities of muscle, cultured cells and isolated mitochondria from a variety of species and tissues, by using a single-wavelength spectrophotometer. An efficient tissue disruption and the choice for each assay of specific buffers, substrates, adjuvants and detergents in a narrow concentration range allow maximal sensitivity, specificity and linearity of the kinetics. This protocol can be completed in 3 h.

Melanoma addiction to the long non-coding RNA SAMMSON

Focal amplifications of chromosome 3p13–3p14 occur in about 10% of melanomas and are associated with a poor prognosis. The melanoma-specific oncogene MITF resides at the epicentre of this amplicon. However, whether other loci present in this amplicon also contribute to melanomagenesis is unknown. Here we show that the recently annotated long non-coding RNA (lncRNA) gene SAMMSON is consistently co-gained with MITF. In addition, SAMMSON is a target of the lineage-specific transcription factor SOX10 and its expression is detectable in more than 90% of human melanomas. Whereas exogenous SAMMSON increases the clonogenic potential in trans, SAMMSON knockdown drastically decreases the viability of melanoma cells irrespective of their transcriptional cell state and BRAF, NRAS or TP53 mutational status. Moreover, SAMMSON targeting sensitizes melanoma to MAPK-targeting therapeutics both in vitro and in patient-derived xenograft models. Mechanistically, SAMMSON interacts with p32, a master regulator of mitochondrial homeostasis and metabolism, to increase its mitochondrial targeting and pro-oncogenic function. Our results indicate that silencing of the lineage addiction oncogene SAMMSON disrupts vital mitochondrial functions in a cancer-cell-specific manner; this silencing is therefore expected to deliver highly effective and tissue-restricted anti-melanoma therapeutic responses.

A novel deletion in the GTPase domain of OPA1 causes defects in mitochondrial morphology and distribution, but not in function

Autosomal dominant optic atrophy (ADOA), the commonest cause of inherited optic atrophy, is caused by mutations in the ubiquitously expressed gene optic atrophy 1 (OPA1), involved in fusion and biogenesis of the inner membrane of mitochondria. Bioenergetic failure, mitochondrial network abnormalities and increased apoptosis have all been proposed as possible causal factors. However, their relative contribution to pathogenesis as well as the prominent susceptibility of the retinal ganglion cell (RGC) in this disease remains uncertain. Here we identify a novel deletion of OPA1 gene in the GTPase domain in three patients affected by ADOA. Muscle biopsy of the patients showed neurogenic atrophy and abnormal morphology and distribution of mitochondria. Confocal microscopy revealed increased mitochondrial fragmentation in fibroblasts as well as in myotubes, where mitochondria were also unevenly distributed, with clustered organelles alternating with areas where mitochondria were sparse. These abnormalities were not associated with altered bioenergetics or increased susceptibility to pro-apoptotic stimuli. Therefore, changes in mitochondrial shape and distribution can be independent of other reported effects of OPA1 mutations, and therefore may be the primary cause of the disease. The arrangement of mitochondria in RGCs, which degenerate in ADOA, may be exquisitely sensitive to disturbance, and this may lead to bioenergetic crisis and/or induction of apoptosis. Our results highlight the importance of mitochondrial dynamics in the disease per se, and point to the loss of the fine positioning of mitochondria in the axons of RGCs as a possible explanation for their predominant degeneration in ADOA.

Phenotypic heterogeneity of the 8344A.G mtDNA "MERRF" mutation

Objectives: Myoclonic epilepsy with ragged-red fibers (MERRF) is a rare mitochondrial syndrome, mostly caused by the 8344A>G mitochondrial DNA mutation. Most of the previous studies have been based on single case/family reports or series with few patients. The primary aim of this study was the characterization of a large cohort of patients with the 8344A>G mutation. The secondary aim was revision of the previously published data. Methods: Retrospective, database-based study (Nation-wide Italian Collaborative Network of Mitochondrial Diseases) and systematic revision. Results: Forty-two patients carrying the mutation were identified. The great majority did not have full-blown MERRF syndrome. Myoclonus was present in 1 of 5 patients, whereas myopathic signs and symptoms, generalized seizures, hearing loss, eyelid ptosis, and multiple lipomatosis represented the most common clinical features. Some asymptomatic mutation carriers have also been observed. Myoclonus was more strictly associated with ataxia than generalized seizures in adult 8344A>G subjects. Considering all of the 321 patients so far available, including our dataset and previously published cases, at the mean age of approximately 35 years, the clinical picture was characterized by the following signs/symptoms, in descending order: myoclonus, muscle weakness, ataxia (35%–45% of patients); generalized seizures, hearing loss (25%–34.9%); cognitive impairment, multiple lipomatosis, neuropathy, exercise intolerance (15%–24.9%); and increased creatine kinase levels, ptosis/ophthalmoparesis, optic atrophy, cardiomyopathy, muscle wasting, respiratory impairment, diabetes, muscle pain, tremor, migraine (5%–14.9%). Conclusions: Our results showed higher clinical heterogeneity than commonly thought. Moreover, MERRF could be better defined as a myoclonic ataxia rather than a myoclonic epilepsy.

Myelo-optico-neuropathy in copper deficiency occurring after partial gastrectomy: Do small bowel bacterial overgrowth syndrome and occult zinc ingestion tip the balance?

Acquired copper deficiency has recently been recognized as a cause of myeloneuropathy mimicking subacute combined degeneration due to vitamin B-12 deficiency. A remote history of gastric surgery is frequently associated with this syndrome. However, the very limited prevalence of severe copper deficiency in patients with a history of gastric surgery suggests that additional contributing factors are likely to be involved. We describe a patient with copper deficiency and a previous Billroth II partial gastrectomy for gastric carcinoma, presenting with severe myelo-optico-neuropathy, demyelinating lesions of the brain, and subjective hyposmia. An abnormal glucose breath test also revealed small bowel bacterial overgrowth syndrome. Copper replacement therapy associated with antibiotic therapy was effective in preventing further neurological damage and in obtaining mild improvement. We propose that copper status should be evaluated in all patients presenting with unexplained noninflammatory myeloneuropathy. Small bowel bacterial overgrowth syndrome should be investigated as a cause of generalized malabsorption and a possible contributing factor to copper deficiency after gastric surgery, as should occult zinc ingestion.

Optimization of respiratory chain enzymatic assays in muscle for the diagnosis of mitochondrial disorders

The diagnosis of mitochondrial disorders is difficult due to clinical and genetic heterogeneity. Measurements of mitochondrial respiratory chain (RC) enzyme activities are essential for both clinical diagnoses and many basic research questions. Current protocols for RC analysis are not standardized, and so are prone to inter-laboratory variability, and also to biochemical interferences that lead to analytical discrepancies. Moreover, knowledge of the analytical performances of these assays, which is essential to draw meaningful conclusions from the results, is lacking. To understand this variability and to propose possible solutions, we systematically investigated the effect of different homogenization protocols and chemical conditions on RC assays using muscle homogenates. We developed optimized protocols and a novel complex III method with improved sensitivity, precision, and linearity. These methods can be reliably performed on minute muscle samples with a single-wavelength spectrophotometer. Moreover, we measured the variability of the proposed homogenization protocol and we provide a systematic evaluation of each assays specificity, precision, and linearity. These data will be useful for quality control in both clinical and research laboratories.

Subacute sensory ataxia and optic neuropathy with thiamine deficiency

Background. A 71 year-old man with a history of partial gastrectomy presented to the emergency department with subacute gait instability associated with painful dysesthesias and clumsiness in both hands. 10 years before presentation he had received a diagnosis of megaloblastic anemia, with no neurological involvement, as a result of vitamin B12 and folate deficiency, for which he was receiving regular supplements.Investigations. Neurological examination; routine laboratory testing; MRI of the spine and brain; lumbar puncture; electromyography; sensory, motor and visual evoked potentials, optic nerve optical coherence tomography; immunoelectrophoresis; cryoglobulins; immunological and infection tests; screening for onconeural antibodies; measurement of serum metabolic values, including vitamins B12 and E, folates, homocysteine, copper, zinc and pyruvic acid; transketolase activity; gastrointestinal endoscopies; and the glucose breath test.Diagnosis. Subacute sensory ataxia with bilateral optic neuropathy related to thiamine deficiency resulting from remote partial gastrectomy.Management. Parenteral thiamine supplementation followed by chronic oral thiamine and short-term, low-dose multivitamins.

Primary coenzyme Q10 deficiency presenting as fatal neonatal multiorgan failure.

Coenzyme Q10 deficiency is a clinically and genetically heterogeneous disorder, with manifestations that may range from fatal neonatal multisystem failure, to adult-onset encephalopathy. We report a patient who presented at birth with severe lactic acidosis, proteinuria, dicarboxylic aciduria, and hepatic insufficiency. She also had dilation of left ventricle on echocardiography. Her neurological condition rapidly worsened and despite aggressive care she died at 23 h of life. Muscle histology displayed lipid accumulation. Electron microscopy showed markedly swollen mitochondria with fragmented cristae. Respiratory-chain enzymatic assays showed a reduction of combined activities of complex I+III and II+III with normal activities of isolated complexes. The defect was confirmed in fibroblasts, where it could be rescued by supplementing the culture medium with 10 μM coenzyme Q10. Coenzyme Q10 levels were reduced (28% of controls) in these cells. We performed exome sequencing and focused the analysis on genes involved in coenzyme Q10 biosynthesis. The patient harbored a homozygous c.545T>G, p.(Met182Arg) alteration in COQ2, which was validated by functional complementation in yeast. In this case the biochemical and morphological features were essential to direct the genetic diagnosis. The parents had another pregnancy after the biochemical diagnosis was established, but before the identification of the genetic defect. Because of the potentially high recurrence risk, and given the importance of early CoQ10 supplementation, we decided to treat with CoQ10 the newborn child pending the results of the biochemical assays. Clinicians should consider a similar management in siblings of patients with CoQ10 deficiency without a genetic diagnosis.

Deficiency of the miR-29a/b-1 cluster leads to ataxic features and cerebellar alterations in mice.

miR-29 is expressed strongly in the brain and alterations in expression have been linked to several neurological disorders. To further explore the function of this miRNA in the brain, we generated miR-29a/b-1 knockout animals. Knockout mice develop a progressive disorder characterized by locomotor impairment and ataxia. The different members of the miR-29 family are strongly expressed in neurons of the olfactory bulb, the hippocampus and in the Purkinje cells of the cerebellum. Morphological analysis showed that Purkinje cells are smaller and display less dendritic arborisation compared to their wildtype littermates. In addition, a decreased number of parallel fibers form synapses on the Purkinje cells. We identified several mRNAs significantly up-regulated in the absence of the miR-29a/b-1 cluster. At the protein level, however, the voltage-gated potassium channel Kcnc3 (Kv3.3) was significantly up-regulated in the cerebella of the miR-29a/b knockout mice. Dysregulation of KCNC3 expression may contribute to the ataxic phenotype.

IMMUNOTHERAPY-REVERSED COMPULSIVE, MONOAMINERGIC, CIRCADIAN RHYTHM DISORDER IN MORVAN SYNDROME

Antibodies to voltage-gated potassium channels (VGKC-Abs) have been recognized in neuromyotonia, limbic encephalitis (LE), and Morvan syndrome (MoS),1,2 a rare condition with insomnia, peripheral, and central and autonomic nervous system involvement. Clinical progression in MoS is variable, but potentially fatal. We describe a patient with VGKC-Abs-positive MoS who showed prominent compulsive behaviors, basal ganglia hypermetabolism, increased catecholamine and serotonin secretion, epileptic seizures, and cardiac and endocrine circadian rhythm suppression. A striking response to immunotherapy paralleled a marked reduction in VGKC-Abs over 15 months. ### Case report. A 64-year-old man with idiopathic pulmonary fibrosis and polyarthrosis developed cramps, lower extremity pain, and sensory loss. Three years later, his sleep was disturbed by chewing, manual stereotypies, and sleep-talking. After an episode of tonic-clonic seizures, EEG revealed a temporal epileptic focus. He developed paroxysmal confusional episodes, with gesturing and irregular breathing, that were unresponsive to multiple anticonvulsants. He became irritable and hypomanic with overwhelming compulsive shopping and stealing. Urinary frequency, anosmia, impotence, and increased appetite appeared. Examination showed bilateral postural tremor, multifocal myoclonus, lower extremity fasciculations, hypoesthesia, hyporeflexia, and mild proximal muscle atrophy. Laboratory investigations, including autoimmune and thyroid profile, were normal except for increased leukocytes, a raised erythrocyte sedimentation rate and creatine phosphokinase, mild hyponatremia, and antinuclear antibodies 1:160. CSF was negative for infections and 14.3.3 protein, but showed slightly increased …