Giovanni Rizzo

  The aim of this study was to evaluate the presence of abnormalities in the brain of patients with restless legs syndrome (RLS) using voxel-based morphometry and diffusion tensor imaging (DTI).   Twenty patients and twenty controls were studied. Voxel-based morphometry analysis was performed using statistical parametric mapping (SPM8) and FSL-VBM software tools. For voxel-wise analysis of DTI, tract-based spatial statistics (TBSS) and SPM8 were used.   Applying an appropriate threshold of probability, no significant results were found either in comparison or in correlation analyses.   Our data argue against clear structural or microstructural abnormalities in the brain of patients with idiopathic RLS, suggesting a prevalent role of functional or metabolic impairment.

Objective Mounting evidence links neurodegenerative disorders such as Parkinson and Alzheimer disease with mitochondrial dysfunction and recent emphasis has focused on mitochondrial dynamics and quality control. Mitochondrial dynamics and mtDNA maintenance is another link emerged recently, implicating mutations in the mitochondrial fusion genes OPA1 and MFN2 in the pathogenesis of multisystem syndromes characterized by neurodegeneration and accumulation of mtDNA multiple deletions in post-mitotic tissues. Here, we report two Italian families affected by dominant chronic progressive external ophthalmoplegia (CPEO) complicated by parkinsonism and dementia. Methods Patients were extensively studied by optical coherence tomography (OCT) to assess retinal nerve fibers, muscle and brain MR spectroscopy, analysis of muscle biopsy and fibroblasts. Candidate genes were sequenced and mtDNA was analyzed for rearrangements. Results Affected individuals displayed a slowly progressive syndrome characterized by CPEO, mitochondrial myopathy, sensorineural deafness, peripheral neuropathy, parkinsonism and/or cognitive impairment, in most cases without visual complains, but with subclinical loss of retinal nerve fibers at OCT. Muscle biopsies showed COX negative fibers and mtDNA multiple deletions, and MR spectroscopy displayed defective oxidative metabolism in muscle and brain. We found two heterozygous OPA1 missense mutations affecting highly conserved amino acid positions (p.G488R, p.A495V) in the GTPase domain, each segregating with affected individuals. Fibroblast studies showed reduced amount of OPA1 protein with normal mRNA expression, fragmented mitochondria, impaired bioenergetics, increased autophagy and mitophagy. Interpretation The association of CPEO and parkinsonism/dementia with subclinical optic neuropathy widens the phenotypic spectrum of OPA1 mutations, highlighting the link of defective mitochondrial dynamics, mtDNA multiple deletions and altered mitophagy with parkinsonism. This article is protected by copyright. All rights reserved.

Neurophysiological investigations disclosed spinal cord hyperexcitability in primary restless legs syndrome (p-RLS). Uremic RLS (u-RLS) is the most common secondary form, but its pathophysiological mechanisms remain unsettled. Aim of this study was to explore spinal cord excitability by evaluating group I nonreciprocal (Ib) inhibition in u-RLS patients in comparison with p-RLS patients and healthy subjects. Eleven u-RLS patients undergoing long-term hemodialysis treatment, nine p-RLS patients and ten healthy subjects were studied. Soleus H reflex latency (HR-L), H(max)/M(max) ratio, and Ib inhibition were evaluated. Ib inhibition was tested measuring the amplitude changes in soleus H reflex following stimulation of the synergist gastrocnemius medialis (GM) nerve at rest. Nerve conduction studies were performed in the uremic patients. The H(max)/M(max) ratio did not differ in the three groups. The u-RLS patients showed a normal Ib inhibition comparable with the healthy group, whereas the p-RLS group had evidence of a reduced active inhibition compared with both u-RLS patients (P = 0.04) and controls (P = 0.007), prominently at 5 ms (P = 0.007) and at 6 ms (P = 0.02) of conditioning-test interval. Neurophysiological examination disclosed abnormalities ranging from higher HR-L to clear-cut polyneuropathy in most u-RLS patients. Unlike p-RLS patients, u-RLS patients had normal Ib inhibition, suggesting a regular supraspinal control of Ib spinal interneurons. Subclinical peripheral nerve abnormalities were detected in most uremic patients. Peripherally disrupted sensory modulation may represent the major pathophysiological determinant of uremic RLS.

To assess the peripapillary retinal nerve fiber and macular retinal ganglion cell (RGC) loss in patients with dominant optic atrophy (DOA) stratified by OPA1 mutation type. Cross-sectional study. We studied 39 patients from 28 pedigrees with DOA harboring heterozygous mutations in the OPA1 gene along with 45 age-matched healthy subjects. The retinal nerve fiber layer (RNFL) and ganglion cell-inner plexiform layer (GC-IPL) of patients with DOA were evaluated by optical coherence tomography (OCT) and compared to those of controls. Patients' eyes were divided into 4 groups based on increasing severity of visual loss (DOA1 to DOA4) and were stratified by OPA1 mutation type. The average thicknesses of the RNFL and GC-IPL were smaller in patients with DOA than in healthy controls (P < 0.0001). RNFL analysis showed a significant reduction of the average, superior and inferior quadrants thicknesses in the DOA4 group compared to the DOA1 group (P = 0.001, P = 0.002 and P = 0.001, respectively). GC-IPL analysis showed a significant thinning in the superotemporal and superior sectors in the patients with DOA2 compared to those with DOA1 (P = 0.046 and P = 0.04, respectively). Stratifying by mutation type, average, superior and nasal RNFL thinning was significantly more severe in missense mutations and had a presumed dominant-negative effect compared to mutations causing haploinsufficiency. The present study demonstrates that in DOA, loss of macular RGCs is the earliest pathologic event, better reflected by GC-IPL measurements, whereas RNFL thickness is a measure of spared axons in late stages of the disease. Thus, mild cases (DOA2) show significant macular RGC loss as opposed to substantial maintenance of RNFL thickness, which is significantly decreased only in severe cases (DOA4). A clear genotype/phenotype correlation emerged, stratifying OCT measures by OPA1 mutation type, missense mutations being the most severe.

Friedreich ataxia (FRDA) is the commonest form of autosomal recessive ataxia. This study aimed to define the extent of the brain damage in FRDA patients and to identify in vivo markers of neurodegeneration, using diffusion-weighted imaging (DWI). We studied 27 FRDA patients and 21 healthy volunteers using a 1.5 T scanner. Axial DW images were obtained and mean diffusivity (MD) maps were generated. Region of interests (ROIs) included medulla, pons, inferior, middle and superior cerebellar peduncles (ICP, SCP, MCP), dentate nucleus, cerebellar white matter, thalamus, caudate, putamen, pallidus, pyramidal tracts at level of posterior limb of internal capsule (PLIC), optic radiations (OR), and corpus callosum. Histograms of MD were generated for all pixels in the whole cerebral hemispheres and infratentorial compartment. Disease severity was assessed by the International Cooperative Ataxia Rating Scale (ICARS). FRDA patients had significantly higher MD values than controls in medulla (P < 0.001), ICP (P < 0.001), MCP (P < 0.01), SCP (P < 0.001), OR (P < 0.001), and at the level of the infratentorial structures such as brainstem (P < 0.01), cerebellar hemispheres (P < 0.01), and especially in the cerebellar vermis (P < 0.001). MD values were strongly correlated with disease duration and ICARS score. Our results showed that DWI is a suitable non-invasive technique to quantify the extent of neurodegeneration in FRDA, that appears more extended than previously reported, showing a microstructural involvement of structures such as OR and MCP.

ABSTRACT Introduction Restless legs syndrome (RLS) is the most common disorder of movement and quiet wakefulness, with a prevalence in the general population between 3% and 9% (1). The pathophysiology of idiopatic RLS is poorly understood. Clinical and pharmacological observations point towards a central role for the dopaminergic system and iron metabolism (2). The potential role of iron metabolism involvement in RLS is indicated primarily by those secondary causes of RLS in which iron insufficiency is clear, but also from some limited pharmacological studies. Several studies showed a relation between low ferritin concentrations and symptoms of the syndrome, especially when ferritin was measured in the cerebrospinal fluid (CSF). Studies on CSF showed decreased ferritin, elevated transferrin and decreased pro-hepcidin (that interacts with the iron transport protein ferroportin on the surface of cells) in patients with RLS. Neuropathological data showed alterations of iron regulatory proteins in neuromelanin cells from brains of patients (2). Reduced brain iron in RLS patients is also suggested by the data of some MR studies that exploit the effect of iron on T2, T2* and T2', although with discrepant results (3-5). A recent technique that measures the phase shifts in gradient-echo images may be a sensitive tool to quantify the iron content of the brain (6). Tissue containing (paramagnetic) iron exhibits a negative phase in complex images compared to immediately adjacent tissue, which will have an increased phase. Our aim was to use phase imaging to study patients with idiopatic RLS. Methods 11 RLS patients (age 54±11, mean ± SD) and 11 healthy volunteers (age 51±18) were studied in a 1.5 T General Electrics Medical Systems (Milwaukee, Wisconsin) Signa Horizon LX whole-body scanner equipped with a quadrature birdcage head coil. Diagnostic criteria of the International Restless Legs Syndrome Study Group (IRLSSG) were applied. The severity of RLS was assessed using the revised IRLSSG rating scale. Anatomical imaging was performed by a T2-weighted (T2W) FSE sequence in an axial oblique plane, using acquisition parameters: α=90º; echo time (TE): 107 ms; repetition time (TR): 5080 ms; square FOV: 24 cm; acq. matrix 320×256; reconstructed in-plane resolution: 0.938 mm; slice thickness; 4 mm w/o gap. # slices variable to cover whole head. NEX: 2. Phase-sensitized images were acquired using a gradient echo sequence, and preserving both real and imaginary channels. Slice locations matched those of the anatomical scan, excluding slices above the central corpus callosum, and below the dentate nucleus. Acquisition parameters: TE/TR: 40/60 ms; acq. matrix 512×256; reconstructed in-plane resolution: 0.938 mm; NEX 2; bandwidth 15.6 kHz; maximum acq. time 7'06". Following the published method (6) data were high pass filtered by multiplication with a filter function in k-space, using tools provided by FSL (FMRIB; U Oxford) and AFNI (NIMH, NIH; Bethesda MD), and a phase map prepared using the filtered data. T2W data were registered onto the gradient echo data using FLIRT (FSL). Regions of interest were selected in two ways: structures known to accumulate iron (dentate and red nucleus, substantia nigra, basal ganglia) were manually segmented using both phase maps and T2W images. Whole brain regions of interest were selected automatically by thresholding T2W data, and excluding pixels whose local filtered phase dispersion exceeded a second threshold (indicating low signal:noise). For whole brain ROIs, the 10 th , 50 th and 90 th percentile of the filtered phase histogram were calculated (Figure 1F), while for local ROIs, 25 th and 50 th percentiles only, as these contained mainly negative phase. Patients and control groups were compared using the Mann-Whitney U test, and correlation with demographic and clinical parameters used the Spearman test.

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