Expanding the differential diagnosis of inherited neuropathies with non-uniform conduction: Andermann syndrome

Uniform conduction slowing has been considered a characteristic of inherited demyelinating neuropathies. We present an 18-year-old girl, born from first cousins, that presented a late motor and psychological development, cerebellar ataxia, facial diplegia, abnormal eye movement, scoliosis, and corpus callosum agenesis, whose compound muscle action potentials were slowed and dispersed. A mutation was found on KCC3 gene, confirming Andermann syndrome, a disease that must be included in the differential diagnosis of inherited neuropathies with non-uniform conduction slowing.

Cosmetic Orbital Inferomedial Decompression: Quantifying the Risk of Diplopia Associated With Extraocular Muscle Dimensions

Purpose: To quantify the risk of new diplopia in inferomedial orbital decompression performed for cosmetic reasons. Methods: Retrospective analysis of 114 patients with Graves orbitopathy who underwent an inferomedial orbital decompression. No patient had diplopia in any of the gaze positions or optic neuropathy. A single coronal slice 9 mm posterior to the lateral orbital rim was employed to quantify the muscular index of the extraocular recti and of the superior complex. A control group of 56 patients imaged for other reasons were also measured. After surgery the oculomotor status of all patients who complained of diplopia and of 51 patients free of diplopia was measured with the prism and cover test in the primary and secondary gaze positions. Results: The rate of new-onset diplopia was 14.0% (16 patients). Eye deviations were confirmed in 14 patients. Of these, 10 had significant strabismus that warranted surgical or prism treatment. Most patients had esotropia associated with small vertical deviations. The size of the medial and inferior recti was significantly associated with the development of diplopia. The estimated odds for the appearance of diplopia in patients with muscle enlargement was 12.76 (medial rectus) and 5.21 (inferior rectus). Small-angle deviations were also detected in 27.4% of patients who did not experience diplopia. Conclusions: Medial and inferior recti enlargement is a strong predictor of new-onset diplopia. A large number of patients who do not report diplopia also present with small-angle deviations. (Ophthal Plast Reconstr Surg 2012;28:204-207)

Effectiveness of low-level laser therapy for patients with carpal tunnel syndrome: design of a randomized single-blinded controlled trial

Background: Carpal tunnel syndrome is the most common neuropathy in the upper extremity, resulting from the compression of the median nerve at wrist level. Clinical studies are essentials to present evidence on therapeutic resources use at early restoration on peripheral nerve functionality. Low-level laser therapy has been widely investigated in researches related to nerve regeneration. Therefore, it is suggested that the effect of low-level laser therapy associated with other conservative rehabilitation techniques may positively affect symptoms and overall hand function in compressive neuropathies such as carpal tunnel syndrome. The aim of this study is to evaluate the effectiveness of low-level laser therapy in addition to orthoses therapy and home orientations in patients with carpal tunnel syndrome. Methods/Design: Patients older than 18 years old will be included, with clinical diagnosis of carpal tunnel syndrome, excluding comorbidies. A physiotherapist will conduct intervention, with a blinding evaluator. Randomization will be applied to allocate the patients in each group: with association or not to low-level laser therapy. All of them will be submitted to orthoses therapy and home orientations. Outcome will be assessed through: pain visual analogic scale, Semmes Weinstein monofilaments (TM) threshold sensibility test, Pinch Gauge T, Boston Carpal Tunnel Questionnaire and two point discrimination test. Discussion: This paper describes the design of a randomized controlled trial, which aim to assess the effectiveness of conservative treatment added to low-level laser therapy for patients with carpal tunnel syndrome. Trial registration: Brazilian Clinical Trials Registry (ReBec) - 75ddtf / Universal Trial Number: U1111-1121-5184

Sural nerve involvement in experimental hypertension: morphology and morphometry in male and female normotensive Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR)

Background: The sural nerve has been widely investigated in experimental models of neuropathies but information about its involvement in hypertension was not yet explored. The aim of the present study was to compare the morphological and morphometric aspects of different segments of the sural nerve in male and female spontaneously hypertensive (SHR) and normotensive Wistar-Kyoto (WKY) rats. Rats aged 20 weeks (N = 6 in each group) were investigated. After arterial pressure and heart rate recordings in anesthetized animals, right and left sural nerves were removed and prepared for epoxy resin embedding and light microscopy. Morphometric analysis was performed with the aid of computer software, and took into consideration the fascicle area and diameter, as well as myelinated fiber number, density, area and diameter. Results: Significant differences were observed for the myelinated fiber number and density, comparing different genders of WKY and SHR. Also, significant differences for the morphological (thickening of the endoneural blood vessel walls and lumen reduction) and morphometric (myelinated fibers diameter and G ratio) parameters of myelinated fibers were identified. Morphological exam of the myelinated fibers suggested the presence of a neuropathy due to hypertension in both SHR genders. Conclusions: These results indicate that hypertension altered important morphometric parameters related to nerve conduction of sural nerve in hypertensive animals. Moreover the comparison between males and females of WKY and SHR allows the conclusion that the morphological and morphometric parameters of sural nerve are not gender related. The morphometric approach confirmed the presence of neuropathy, mainly associated to the small myelinated fibers. In conclusion, the present study collected evidences that the high blood pressure in SHR is affecting the sural nerve myelinated fibers.

Mutation Spectrum in the Large GTPase Dynamin 2, and Genotype-Phenotype Correlation in Autosomal Dominant Centronuclear Myopathy

Centronuclear myopathy (CNM) is a genetically heterogeneous disorder associated with general skeletal muscle weakness, type I fiber predominance and atrophy, and abnormally centralized nuclei. Autosomal dominant CNM is due to mutations in the large GTPase dynamin 2 (DNM2), a mechanochemical enzyme regulating cytoskeleton and membrane trafficking in cells. To date, 40 families with CNM-related DNM2 mutations have been described, and here we report 60 additional families encompassing a broad genotypic and phenotypic spectrum. In total, 18 different mutations are reported in 100 families and our cohort harbors nine known and four new mutations, including the first splice-site mutation. Genotype-phenotype correlation hypotheses are drawn from the published and new data, and allow an efficient screening strategy for molecular diagnosis. In addition to CNM, dissimilar DNM2 mutations are associated with Charcot-Marie-Tooth (CMT) peripheral neuropathy (CMTD1B and CMT2M), suggesting a tissue-specific impact of the mutations. In this study, we discuss the possible clinical overlap of CNM and CMT, and the biological significance of the respective mutations based on the known functions of dynamin 2 and its protein structure. Defects in membrane trafficking due to DNM2 mutations potentially represent a common pathological mechanism in CNM and CMT. Hum Mutat 33: 949-959, 2012. (C) 2012 Wiley Periodicals, Inc.

Radiation therapy for Graves' ophthalmopathy: a systematic review and meta-analysis of randomized controlled trials

PURPOSE: To evaluate the efficacy of radiotherapy (RT) with total dose of 20 Gy (RT 20 Gy) in the treatment of Graves' ophthalmopathy. METHODS: A systematic review and meta-analysis of randomized controlled trials was performed comparing RT 20 Gy with or without glucocorticoid to clinical treatments for Graves' ophthalmopathy. The MEDLINE, EMBASE, Cochrane Library databases and recent relevant journals were searched. Relevant reports were reviewed by two reviewers. Response to radiotherapy was defined as clinical success according to each trial. We also evaluated the quality of life and whether RT to produce fewer side effects than other treatments. RESULTS: A total of 8 randomized controlled trials (439 patients) were identified. In the subgroup analysis, the overall response to treatment rates was better for: RT 20 Gy plus glucocorticoid vs glucocorticoids alone, OR=17.5 (CI95% 1.85-250, p=0.04), RT 20 Gy vs sham RT, OR= 3.15 (CI95%1.59-6.23, p=0.003) and RT 20Gy plus intravenous glucocorticoid vs RT 20Gy plus oral glucocorticoid, OR=4.15(CI95% 1.34-12.87, p=0.01). There were no differences between RT 20 Gy versus other fractionations and RT 20 Gy versus glucocorticoid alone. RT 20 Gy with or without glucocorticoids showed an improvement in diplopia grade, visual acuity, optic neuropathy, lid width, proptosis and ocular motility. No difference was seen for costs, intraocular pressure and quality of life. CONCLUSION: Our data have shown that RT 20 Gy should be offered as a valid therapeutic option to patients with moderate to severe ophthalmopathy. The effectiveness of orbital radiotherapy can be increased by the synergistic interaction with glucocorticoids. Moreover, RT 20 Gy is useful to improve a lot of ocular symptoms, excluding intraocular pressure, without any difference in quality of life and costs.

Estudo prospectivo da sensibilidade tátil em mãos de uma população brasileira usando pressure-specified sensory device

OBJETIVO: Avaliar os limiares de percepção da pressão em polpas de dois dedos (indicador e mínimo), em uma população brasileira, sem lesão nervosa ou neuropatia. MÉTODOS: Usamos Pressure-Specified Sensory Device, um equipamento computadorizado para obter limiares de percepção da pressão normal, tanto estáticos quanto dinâmicos, e discriminação de dois pontos. RESULTADOS: Testamos a sensibilidade nos dedos, em 30 voluntários. Os testes de significância foram realizados utilizando o teste t de Student. Os valores médios (g/mm²) para os limiares de pressão estática de um e dois pontos (s1PD, s2PD) e discriminação dinâmica de um e dois pontos (m1PD, m2PD) no dedo indicador dominante foram: s1PD = 0,4, m1PD = 0,4, s2PD = 0,48, m2PD = 0,51. CONCLUSÃO: Não há diferença significativa na sensibilidade entre as mãos dominante e não dominante.

Uso da corrente catódica de alta voltagem sobre a dor em compressão nervosa experimental

OBJETIVO: Avaliar o efeito da corrente catódica de alta voltagem sobre a dor em um modelo experimental de ciatalgia. MÉTODOS: Foram utilizados 16 ratos Wistar, machos, submetidos a um modelo de ciatalgia experimental no membro pélvico direito. Os sujeitos foram divididos em grupo simulacro (GS) e grupo tratado com corrente catódica (GP-) por 20 min diários durante 10 dias. O modelo de compressão foi realizado com amarria por fio catgut 4.0 cromado, em quatro pontos ao longo do nervo isquiático. A avaliação da nocicepção foi realizada, de forma funcional, com o tempo de elevação da pata (TEP), e à pressão, pelo limiar de retirada, via analgesímetro eletrônico. Os dados foram coletados antes do modelo de ciatalgia (AV1), três dias depois da compressão (antes, AV2, e após o tratamento, AV3), após o quinto dia de tratamento (AV4) e em seguida ao décimo dia de tratamento (AV5). RESULTADOS: Pela avaliação funcional, em ambos os grupos houve aumento da nocicepção, sem redução da mesma em qualquer momento da avaliação. À pressão, no entanto, o GS mostrou redução do limiar de retirada em todos os momentos, enquanto o GP- apresentou redução do limiar apenas inicialmente - em AV5 o limiar foi restaurado. CONCLUSÃO: Não houve alteração na nocicepção pela avaliação funcional; porém, à pressão, o tratamento com corrente catódica mostrou efeito com a somatória de terapias.

Mitochondrial dysfunction in hereditary optic neuropathies

MITOCHONDRIAL DYSFUNCTION IN HEREDITARY OPTIC NEUROPATHIES Mitochondrial pathologies are a heterogeneous group of clinical manifestations characterized by oxidative phosphorylation impairment. At the beginning of their recognition mitochondrial pathologies were regarded as rare disorders but indeed they are more frequent than originally thought. Due to the unique mitochondria peculiarities mitochondrial pathologies can be caused by mutations in both mitochondrial and nuclear genomes. The poor knowledge of pathologic mechanism of these disorders has not allowed a real development of the “mitochondrial medicine”, that is currently limited to symptoms mitigation. Leber hereditary optic neuropathy (LHON) was the first pathology to be linked to a point mutation in the mtDNA. The mechanism by which point mutations in mitochondrial gene encoding Complex I subunits leads to optic nerve degeneration is still unknown, although is well accepted that other genetic or environmental factors are involved in the modulation of pathology, where a pivotal role is certainly played by oxidative stress. We studied the relationship between the Ala16Val dimorphism in the mitochondrial targeting sequence of nuclear gene SOD2 and the 3460/ND1 LHON mutation. Our results show that, in control population, the heterozygous SOD2 genotype is associated to a higher activity and quantity of MnSOD, particularly with respect to Val homozygotes. Furthermore, we demonstrated that LHON patients harboring at least one Ala allele are characterized by an increased MnSOD activity with respect to relative control population. Since the ATP synthesis rate – severely reduced in LHON patients lymphocytes - is not affected by the SOD2 genotype, we concluded that SOD2 gene could modulate the pathogenicity of LHON mutations through a mechanism associated to an increase of reactive oxygen species production. Autosomal dominant optic atrophy (ADOA) is a pathology linked to mutations in nuclear gene encoding Opa1, a dynamin-related protein localized in the mitochondrial matrix. Although the clinical course is slightly different, the endpoint of ADOA is exactly the same of LHON: optic nerve degeneration with specific involvement of retinal ganglion cells. Opa1 is a relatively new protein, whose major role is the regulation of mitochondrial fusion. Mitochondrial morphology is the results of the equilibrium between two opposite force: fusion and fission, two processes that have to be finely regulated in order to preserve mitochondrial and cellular physiology. We studied fibroblasts deriving from ADOA patients characterized by a new deletion in the GTPase domain of the OPA1 gene. The biochemical characterization of ADOA and control fibroblasts has concerned the evaluation of ATP synthesis rate, mitochondrial membrane potential in different metabolic conditions and the morphological status of mitochondria. Regarding ATP synthesis rate we did not find significant differences between ADOA and control fibroblasts even though a trend toward increased reduction in ADOA samples is observed when fibroblasts are grown in absence of glucose or in the medium containing gramicidin. Furthermore, we found that also in ADOA fibroblasts membrane potential is actively maintained by proton pumping of fully functional respiratory chain complexes. Our results indicate that the mutation found in the pedigree analyzed acts primary impairing the mitochondrial fusion without affecting the energy production, supporting the notion that cell function is tightly linked to mitochondrial morphology. Mitochondrial dysfunctions are acquiring great attention because of their recognized relevance not only in aging but also in age-related pathologies including cancer, cardiovascular disease, type II diabetes, and neurodegenerative disorders. The involvement of mitochondria in such detrimental pathologies that, currently, have become so common enhances the necessity of standardization of therapeutic strategies capable of rescuing the normal mitochondrial function. In order to propose an alternative treatment for energy deficiency-disorders we tested the effect of substrates capable to stimulate the substrate-level phosphorylation on viability and energy availability in different experimental models grown under different metabolic conditions. In fibroblasts, the energy defect was achieved by culturing cells in presence of oligomycin, an inhibitor of ATP synthase complex. NARP cybrids have been used as model of mitochondrial pathology. Cell viability and ATP content have been considered as parameters to assay the capability of exogenous substrate to rescue energy failure. Our results suggest that patients suffering for some forms of ATP synthase deficiency, or characterized by a deficiency in energy production, might benefit from dietary or pharmacological treatment based on supplementation of α-ketoglutarate and aspartate.

Studies of OPA1 pathogenic mechanisms in Dominant Optic Atrophy and novel protein function in mitochondrial DNA stability

The mitochondrion is an essential cytoplasmic organelle that provides most of the energy necessary for eukaryotic cell physiology. Mitochondrial structure and functions are maintained by proteins of both mitochondrial and nuclear origin. These organelles are organized in an extended network that dynamically fuses and divides. Mitochondrial morphology results from the equilibrium between fusion and fission processes, controlled by a family of “mitochondria-shaping” proteins. It is becoming clear that defects in mitochondrial dynamics can impair mitochondrial respiration, morphology and motility, leading to apoptotic cell death in vitro and more or less severe neurodegenerative disorders in vivo in humans. Mutations in OPA1, a nuclear encoded mitochondrial protein, cause autosomal Dominant Optic Atrophy (DOA), a heterogeneous blinding disease characterized by retinal ganglion cell degeneration leading to optic neuropathy (Delettre et al., 2000; Alexander et al., 2000). OPA1 is a mitochondrial dynamin-related guanosine triphosphatase (GTPase) protein involved in mitochondrial network dynamics, cytochrome c storage and apoptosis. This protein is anchored or associated on the inner mitochondrial membrane facing the intermembrane space. Eight OPA1 isoforms resulting from alternative splicing combinations of exon 4, 4b and 5b have been described (Delettre et al., 2001). These variants greatly vary among diverse organs and the presence of specific isoforms has been associated with various mitochondrial functions. The different spliced exons encode domains included in the amino-terminal region and contribute to determine OPA1 functions (Olichon et al., 2006). It has been shown that exon 4, that is conserved throughout evolution, confers functions to OPA1 involved in maintenance of the mitochondrial membrane potential and in the fusion of the network. Conversely, exon 4b and exon 5b, which are vertebrate specific, are involved in regulation of cytochrome c release from mitochondria, and activation of apoptosis, a process restricted to vertebrates (Olichon et al., 2007). While Mgm1p has been identified thanks to its role in mtDNA maintenance, it is only recently that OPA1 has been linked to mtDNA stability. Missense mutations in OPA1 cause accumulation of multiple deletions in skeletal muscle. The syndrome associated to these mutations (DOA-1 plus) is complex, consisting of a combination of dominant optic atrophy, progressive external ophtalmoplegia, peripheral neuropathy, ataxia and deafness (Amati- Bonneau et al., 2008; Hudson et al., 2008). OPA1 is the fifth gene associated with mtDNA “breakage syndrome” together with ANT1, PolG1-2 and TYMP (Spinazzola et al., 2009). In this thesis we show for the first time that specific OPA1 isoforms associated to exon 4b are important for mtDNA stability, by anchoring the nucleoids to the inner mitochondrial membrane. Our results clearly demonstrate that OPA1 isoforms including exon 4b are intimately associated to the maintenance of the mitochondrial genome, as their silencing leads to mtDNA depletion. The mechanism leading to mtDNA loss is associated with replication inhibition in cells where exon 4b containing isoforms were down-regulated. Furthermore silencing of exon 4b associated isoforms is responsible for alteration in mtDNA-nucleoids distribution in the mitochondrial network. In this study it was evidenced that OPA1 exon 4b isoform is cleaved to provide a 10kd peptide embedded in the inner membrane by a second transmembrane domain, that seems to be crucial for mitochondrial genome maintenance and does correspond to the second transmembrane domain of the yeasts orthologue encoded by MGM1 or Msp1, which is also mandatory for this process (Diot et al., 2009; Herlan et al., 2003). Furthermore in this thesis we show that the NT-OPA1-exon 4b peptide co-immuno-precipitates with mtDNA and specifically interacts with two major components of the mitochondrial nucleoids: the polymerase gamma and Tfam. Thus, from these experiments the conclusion is that NT-OPA1- exon 4b peptide contributes to the nucleoid anchoring in the inner mitochondrial membrane, a process that is required for the initiation of mtDNA replication and for the distribution of nucleoids along the network. These data provide new crucial insights in understanding the mechanism involved in maintenance of mtDNA integrity, because they clearly demonstrate that, besides genes implicated in mtDNA replications (i.e. polymerase gamma, Tfam, twinkle and genes involved in the nucleotide pool metabolism), OPA1 and mitochondrial membrane dynamics play also an important role. Noticeably, the effect on mtDNA is different depending on the specific OPA1 isoforms down-regulated, suggesting the involvement of two different combined mechanisms. Over two hundred OPA1 mutations, spread throughout the coding region of the gene, have been described to date, including substitutions, deletions or insertions. Some mutations are predicted to generate a truncated protein inducing haploinsufficiency, whereas the missense nucleotide substitutions result in aminoacidic changes which affect conserved positions of the OPA1 protein. So far, the functional consequences of OPA1 mutations in cells from DOA patients are poorly understood. Phosphorus MR spectroscopy in patients with the c.2708delTTAG deletion revealed a defect in oxidative phosphorylation in muscles (Lodi et al., 2004). An energetic impairment has been also show in fibroblasts with the severe OPA1 R445H mutation (Amati-Bonneau et al., 2005). It has been previously reported by our group that OPA1 mutations leading to haploinsufficiency are associated in fibroblasts to an oxidative phosphorylation dysfunction, mainly involving the respiratory complex I (Zanna et al., 2008). In this study we have evaluated the energetic efficiency of a panel of skin fibroblasts derived from DOA patients, five fibroblast cell lines with OPA1 mutations causing haploinsufficiency (DOA-H) and two cell lines bearing mis-sense aminoacidic substitutions (DOA-AA), and compared with control fibroblasts. Although both types of DOA fibroblasts maintained a similar ATP content when incubated in a glucose-free medium, i.e. when forced to utilize the oxidative phosphorylation only to produce ATP, the mitochondrial ATP synthesis through complex I, measured in digitonin-permeabilized cells, was significantly reduced in cells with OPA1 haploinsufficiency only, whereas it was similar to controls in cells with the missense substitutions. Furthermore, evaluation of the mitochondrial membrane potential (DYm) in the two fibroblast lines DOA-AA and in two DOA-H fibroblasts, namely those bearing the c.2819-2A>C mutation and the c.2708delTTAG microdeletion, revealed an anomalous depolarizing response to oligomycin in DOA-H cell lines only. This finding clearly supports the hypothesis that these mutations cause a significant alteration in the respiratory chain function, which can be unmasked only when the operation of the ATP synthase is prevented. Noticeably, oligomycin-induced depolarization in these cells was almost completely prevented by preincubation with cyclosporin A, a well known inhibitor of the permeability transition pore (PTP). This results is very important because it suggests for the first time that the voltage threshold for PTP opening is altered in DOA-H fibroblasts. Although this issue has not yet been addressed in the present study, several are the mechanisms that have been proposed to lead to PTP deregulation, including in particular increased reactive oxygen species production and alteration of Ca2+ homeostasis, whose role in DOA fibroblasts PTP opening is currently under investigation. Identification of the mechanisms leading to altered threshold for PTP regulation will help our understanding of the pathophysiology of DOA, but also provide a strategy for therapeutic intervention.

Pathogenic mechanisms in mitochondrial optic neuropathies

Leber’s hereditary optic neuropathy (LHON) and Autosomal Dominant Optic Atrophy (ADOA) are the two most common inherited optic neuropathies and both are the result of mitochondrial dysfunctions. Despite the primary mutations causing these disorders are different, being an mtDNA mutation in subunits of complex I in LHON and defects in the nuclear gene encoding the mitochondrial protein OPA1 in ADOA, both pathologies share some peculiar features, such a variable penetrance and tissue-specificity of the pathological processes. Probably, one of the most interesting and unclear aspect of LHON is the variable penetrance. This phenomenon is common in LHON families, most of them being homoplasmic mutant. Inter-family variability of penetrance may be caused by nuclear or mitochondrial ‘secondary’ genetic determinants or other predisposing triggering factors. We identified a compensatory mechanism in LHON patients, able to distinguish affected individuals from unaffected mutation carriers. In fact, carrier individuals resulted more efficient than affected subjects in increasing the mitochondrial biogenesis to compensate for the energetic defect. Thus, the activation of the mitochondrial biogenesis may be a crucial factor in modulating penetrance, determining the fate of subjects harbouring LHON mutations. Furthermore, mtDNA content can be used as a molecular biomarker which, for the first time, clearly differentiates LHON affected from LHON carrier individuals, providing a valid mechanism that may be exploited for development of therapeutic strategies. Although the mitochondrial biogenesis gained a relevant role in LHON pathogenesis, we failed to identify a genetic modifying factor for the variable penetrance in a set of candidate genes involved in the regulation of this process. A more systematic high-throughput approach will be necessary to select the genetic variants responsible for the different efficiency in activating mitochondrial biogenesis. A genetic modifying factor was instead identified in the MnSOD gene. The SNP Ala16Val in this gene seems to modulate LHON penetrance, since the Ala allele in this position significantly predisposes to be affected. Thus, we propose that high MnSOD activity in mitochondria of LHON subjects may produce an overload of H2O2 for the antioxidant machinery, leading to release from mitochondria of this radical and promoting a severe cell damage and death ADOA is due to mutation in the OPA1 gene in the large majority of cases. The causative nuclear defects in the remaining families with DOA have not been identified yet, but a small number of families have been mapped to other chromosomal loci (OPA3, OPA4, OPA5, OPA7, OPA8). Recently, a form of DOA and premature cataract (ADOAC) has been associated to pathogenic mutations of the OPA3 gene, encoding a mitochondrial protein. In the last year OPA3 has been investigated by two different groups, but a clear function for this protein and the pathogenic mechanism leading to ADOAC are still unclear. Our study on OPA3 provides new information about the pattern of expression of the two isoforms OPA3V1 and OPA3V2, and, moreover, suggests that OPA3 may have a different function in mitochondria from OPA1, the major site for ADOA mutations. In fact, based on our results, we propose that OPA3 is not involved in the mitochondrial fusion process, but, on the contrary, it may regulate mitochondrial fission. Furthermore, at difference from OPA1, we excluded a role for OPA3 in mtDNA maintenance and we failed to identify a direct interaction between OPA3 and OPA1. Considering the results from overexpression and silencing of OPA3, we can conclude that the overexpression has more drastic consequences on the cells than silencing, suggesting that OPA3 may cause optic atrophy via a gain-of-function mechanism. These data provide a new starting point for future investigations aimed at identifying the exact function of OPA3 and the pathogenic mechanism causing ADOAC.

Il coniglio come modello sperimentale per lo studio degli effetti neurologici indotti da Cobalto

Sono stati studiati gli effetti tossici dell’esposizione cronica a cobalto e cromo. In passato, questa tossicità, che colpiva lavoratori esposti per ragioni occupazionali, è stata un problema molto sentito. Tuttavia, recenti pubblicazioni hanno descritto una specifica tossicità mediata da elevati livelli di cobalto e cromo, anche in pazienti portatori di protesi metalliche, quali gli impianti d’anca. Anche se sintomi clinici tra cui, cecità, sordità e neuropatia periferica, suggeriscono uno specifico neurotropismo, ancora poco è conosciuto delle basi neuropatologiche di questo processo ed oltretutto non ne è ancora stata apportata un’evidenza sperimentale. In questo progetto di ricerca, quindi, si è voluto approfondire il meccanismo patogenetico da cui scaturiscono tali sintomi neurologici, utilizzando come modello sperimentale il coniglio. Conigli New Zealand White sono stati trattati con dosi endovenose ripetute di cobalto e cromo, inoculati singolarmente od in associazione tra loro. Nessuna evidente alterazione clinica o patologica è stata associata alla somministrazione di solo cromo, nonostante gli elevati livelli in sangue e tessuti, mentre i trattati con cobalto-cromo o solo cobalto hanno mostrato segni clinici gravanti sul sistema vestibolo-cocleare; il cobalto, quindi, è stato identificato come il maggiore elemento scatenante neurotossicità. Inoltre all’esame istopatologico gli animali hanno mostrato severa deplezione delle cellule gangliari retiniche e cocleari, assieme a danno al nervo ottico e perdita di cellule sensitive capellute dell’orecchio. È risultato infine evidente che la gravità delle alterazioni è stata correlata al dosaggio ed al tempo di esposizione; dati questi che confermano, quindi, le precedenti osservazioni fatte su pazienti umani esposti a rilascio abnorme di cobalto e cromo da usura di protesi d’anca. È stato ipotizzato che il cobalto agisca sui mitocondri provocando l’incremento di produzione di specie reattive dell’ossigeno e il rilascio di fattori proapoptotici, causando sulle cellule neuronali un danno proporzionale al loro fabbisogno energetico e grado di mielinizzazione.

Melanopsin Retinal Ganglion Cells: relevance to circadian rhythms and sleep in neurodegeneration

In this PhD thesis 3 projects were addressed focusing on the melanopsin retinal ganglion cells (mRGCs) system and its relevance for circadian rhythms and sleep in neurodegeneration. The first project was aimed at completing the characterization of mRGCs system in hereditary optic neuropathies (LHON and DOA). We confirmed that mRGCs are relatively spared also in post-mortem retinal specimens of a DOA case and pupillometric evaluation of LHON patients showed preservation of the pupillary light reflex, with attenuated responses compared to controls. Cell studies failed to indicate a protective role exerted by melanopsin itself. The second project was aimed at characterizing the possible occurrence of optic neuropathy and rest-activity circadian rhythm dysfunction in Alzheimer (AD) and Parkinson disease (PD), as well as, at histological level, the possible involvement of mRGCs in AD. OCT studies demonstrated a subclinical optic neuropathy in both AD and PD patients, with a different pattern involving the superior and nasal quadrants in AD and the temporal quadrant in PD. Actigraphic studies demonstrated a tendency towards an increased intradaily variability (IV) and reduced relative amplitude (RA) of rest-activity circadian rhythm in AD and a significant increased IV a reduced RA in PD. Immunohistochemical analysis of post-mortem retinal specimens and optic nerve cross-sections of neuropathologically confirmed AD cases demonstrated a significant loss of mRGCs and a nearly significant loss of axons in AD compared to controls. The mRGCs were affected in AD independently from age and magnitude of axonal loss. Overall these results suggest a role of the mRGCs system in the pathogenesis of circadian dysfunction in AD. The third project was aimed at evaluating the possible association between a single nucleotide polymorphism of the OPN4 gene and chronotype or SAD, failing to find any significant association with chronotype, but showing a non-significant increment of TT genotype in SAD.

Relationship between pain and anxiety in rats

Clinically, it is well known that neuropathic pain often induces comorbid symptoms such as anxiety. In turn, also anxiety has been associated with a heightened experience of pain. Although, the link between pain and anxiety is well recognized in humans, the neurobiological basis of this relationship remains unclear. Therefore, the aim of the current study was to investigate the influence of neuropathic pain on anxiety and vice versa in rats by assessing not only pain-related behaviour but also by discovering possible key substrates which are responsible for the interrelation of pain and anxiety.rnIn rats with a chronic constriction of the sciatic nerve (CCI model) anxiety-like behaviour was observed. Since anxiety behaviour could be completely abolished after the treatment of the pure analgesic drugs gabapentin and morphine, we concluded that anxiety was caused by the strong persistent pain. Furthermore, we found that the neuropeptides oxytocin and vasopressin were upregulated in the amygdala of CCI rats, and the intra-amygdala treatment of an oxytocin antagonist but not the vasopressin antagonist could reduce anxiety-like behaviour in these animals, while no effect on mechanical hypersensitivity was observed. These data indicate that oxytocin is implicated in the underlying neuronal processes of pain-induced anxiety and helps to elucidate the pathophysiological mechanisms of neuropathic pain. rnTo assess the influence of trait anxiety on pain sensation in rats, we determined mechanical hypersensitivity after sciatic nerve lesion (CCI) in animals selectively bred for high anxiety or low anxiety behaviour. The paw withdrawal thresholds were significantly decreased in high anxiety animals in comparison to low anxiety animals 2 and 3 weeks after surgery. In a second model state anxiety was induced by the sub-chronic injection of the anxiogenic drug pentylentetrazol in naive rats. Pain response to mechanical stimuli was increased after pharmacologically-induced anxiety. These results provided evidence for the influence of both trait and state anxiety on pain sensation. rnThe studies contribute to the elucidation of the relationship between pain and anxiety. We investigated that the neuropathic pain model displays sensory as well as emotional factors of peripheral neuropathy. Changes in expression levels of neuropeptides in the central nervous system due to neuropathic pain may contribute to the pathophysiology of neuropathic pain and its related symptoms in animals which might also be relevant for human scenarios. The results of the current study also confirm that anxiety plays an important role in the perception of pain. rnA better understanding of pain behaviour in animals might improve the preclinical profiling of analgesic drugs during development. The study highlights the potential use of the rat model as a new preclinical tool to further investigate the link between pain and anxiety by determining not only the sensory reflexes after painful stimuli but also the more complex pain-related behaviour such as anxiety.rn

Immunomodulatory therapy to achieve maximum efficacy: doses, monitoring, compliance, and self-infusion at home

The Oxford Programme for Immunomodulatory Immunoglobulin Therapy has been operating since 1992 at Oxford Radcliffe Hospitals in the UK. Initially, this program was set up for patients with multifocal motor neuropathy or chronic inflammatory demyelinating poly-neuropathy to receive reduced doses of intravenous immunoglobulin (IVIG) in clinic on a regular basis (usually every 3 weeks). The program then rapidly expanded to include self-infusion at home, which monitoring showed to be safe and effective. It has been since extended to the treatment of other autoimmune diseases in which IVIG has been shown to be efficacious.