"Build Your Village"-Conducting the Village Test on Cognitively Impaired Patients: A First Journey into Alzheimerland.

BACKGROUND: This work aimed to study the Village Test (VT) in a group of patients with Alzheimer's disease (AD) and compare the results with those of a group of patients with mild cognitive impairment (MCI) and controls. METHODS: A total of 50 patients with AD, 28 patients with MCI, and 38 controls were evaluated. All participants underwent the VT and an extensive neuropsychological evaluation. RESULTS: The mean ages of the participants were 74.4 years for those with AD, 74 for those with MCI, and 70.2 for the controls. The AD group built smaller and essential villages with a scarce use of pieces, a poor use of dynamic pieces, and scarce use of human figures. All constructions were often concentrated in the center of the table. CONCLUSIONS: The villages built by the AD group represent a cognitive and affective coarctation and indicate a sense of existential disorientation and isolation. The VT is a useful aid for getting in touch with the inner emotional and existential states of patients with AD, and it could represent a complementary screening tool for orienting cognitive impairment diagnoses.

Co-occurrence of glial fibrillary acidic protein astrocytopathy in a patient with Leber's hereditary optic neuropathy due to DNAJC30 mutations.

Leber's hereditary optic neuropathy (LHON) is a mitochondrial disease characterized by visual loss, and rarely associated with extraocular manifestations including multiple sclerosis-like lesions. The association of LHON and neuromyelitis optica spectrum disorders has rarely been reported. Here is reported a case of glial fibrillary acidic protein astrocytopathy presenting with area postrema syndrome in a patient with previously diagnosed recessive LHON due to mutations in the nuclear gene DNAJC30. This case emphasizes the necessity of extensive investigations for other treatable conditions in patients with LHON and otherwise unexplained extraocular involvement and the possibility that also visual symptoms can respond to immune therapy.

Deoxyguanosine kinase deficiency: natural history and liver transplant outcome.

Autosomal recessive pathogenetic variants in the DGUOK gene cause deficiency of deoxyguanosine kinase activity and mitochondrial deoxynucleotides pool imbalance, consequently, leading to quantitative and/or qualitative impairment of mitochondrial DNA synthesis. Typically, patients present early-onset liver failure with or without neurological involvement and a clinical course rapidly progressing to death. This is an international multicentre study aiming to provide a retrospective natural history of deoxyguanosine kinase deficient patients. A systematic literature review from January 2001 to June 2023 was conducted. Physicians of research centres or clinicians all around the world caring for previously reported patients were contacted to provide followup information or additional clinical, biochemical, histological/histochemical, and molecular genetics data for unreported cases with a confirmed molecular diagnosis of deoxyguanosine kinase deficiency. A cohort of 202 genetically confirmed patients, 36 unreported, and 166 from a systematic literature review, were analyzed. Patients had a neonatal onset (≤ 1 month) in 55.7% of cases, infantile (>1 month and ≤ 1 year) in 32.3%, pediatric (>1 year and ≤18 years) in 2.5% and adult (>18 years) in 9.5%. Kaplan-Meier analysis showed statistically different survival rates (P < 0.0001) among the four age groups with the highest mortality for neonatal onset. Based on the clinical phenotype, we defined four different clinical subtypes: hepatocerebral (58.8%), isolated hepatopathy (21.9%), hepatomyoencephalopathy (9.6%), and isolated myopathy (9.6%). Muscle involvement was predominant in adult-onset cases whereas liver dysfunction causes morbidity and mortality in early-onset patients with a median survival of less than 1 year. No genotype-phenotype correlation was identified. Liver transplant significantly modified the survival rate in 26 treated patients when compared with untreated. Only six patients had additional mild neurological signs after liver transplant. In conclusion, deoxyguanosine kinase deficiency is a disease spectrum with a prevalent liver and brain tissue specificity in neonatal and infantile-onset patients and muscle tissue specificity in adult-onset cases. Our study provides clinical, molecular genetics and biochemical data for early diagnosis, clinical trial planning and immediate intervention with liver transplant and/or nucleoside supplementation.

Digenic Leigh syndrome on the background of the m.11778G>A Leber hereditary optic neuropathy variant.

Leigh syndrome spectrum (LSS) is a primary mitochondrial disorder defined neuropathologically by a subacute necrotizing encephalomyelopathy and characterized by bilateral basal ganglia and/or brainstem lesions. LSS is associated with variants in several mitochondrial DNA genes and more than 100 nuclear genes, most often related to mitochondrial complex I (CI) dysfunction. Rarely, LSS has been reported in association with primary Leber hereditary optic neuropathy (LHON) variants of the mitochondrial DNA, coding for CI subunits (m.3460G>A in MT-ND1, m.11778G>A in MT-ND4 and m.14484T>C in MT-ND6). The underlying mechanism by which these variants manifest as LSS, a severe neurodegenerative disease, as opposed to the LHON phenotype of isolated optic neuropathy, remains an open question. Here, we analyse the exome sequencing of six probands with LSS carrying primary LHON variants, and report digenic co-occurrence of the m.11778G > A variant with damaging heterozygous variants in nuclear disease genes encoding CI subunits as a plausible explanation. Our findings suggest a digenic mechanism of disease for m.11778G>A-associated LSS, consistent with recent reports of digenic disease in individuals manifesting with LSS due to biallelic variants in the recessive LHON-associated disease gene DNAJC30 in combination with heterozygous variants in CI subunits.

Therapeutic benefit of idebenone in patients with Leber hereditary optic neuropathy: The LEROS nonrandomized controlled trial.

Leber hereditary optic neuropathy (LHON) is a mitochondrial disease leading to rapid and severe bilateral vision loss. Idebenone has been shown to be effective in stabilizing and restoring vision in patients treated within 1 year of onset of vision loss. The open-label, international, multicenter, natural history-controlled LEROS study (ClinicalTrials.gov NCT02774005) assesses the efficacy and safety of idebenone treatment (900 mg/day) in patients with LHON up to 5 years after symptom onset (N = 199) and over a treatment period of 24 months, compared to an external natural history control cohort (N = 372), matched by time since symptom onset. LEROS meets its primary endpoint and confirms the long-term efficacy of idebenone in the subacute/dynamic and chronic phases; the treatment effect varies depending on disease phase and the causative mtDNA mutation. The findings of the LEROS study will help guide the clinical management of patients with LHON.

Bi-allelic variants in SNF8 cause a disease spectrum ranging from severe developmental and epileptic encephalopathy to syndromic optic atrophy.

The endosomal sorting complex required for transport (ESCRT) machinery is essential for membrane remodeling and autophagy and it comprises three multi-subunit complexes (ESCRT I-III). We report nine individuals from six families presenting with a spectrum of neurodevelopmental/neurodegenerative features caused by bi-allelic variants in SNF8 (GenBank: NM_007241.4), encoding the ESCRT-II subunit SNF8. The phenotypic spectrum included four individuals with severe developmental and epileptic encephalopathy, massive reduction of white matter, hypo-/aplasia of the corpus callosum, neurodevelopmental arrest, and early death. A second cohort shows a milder phenotype with intellectual disability, childhood-onset optic atrophy, or ataxia. All mildly affected individuals shared the same hypomorphic variant, c.304G>A (p.Val102Ile). In patient-derived fibroblasts, bi-allelic SNF8 variants cause loss of ESCRT-II subunits. Snf8 loss of function in zebrafish results in global developmental delay and altered embryo morphology, impaired optic nerve development, and reduced forebrain size. In vivo experiments corroborated the pathogenicity of the tested SNF8 variants and their variable impact on embryo development, validating the observed clinical heterogeneity. Taken together, we conclude that loss of ESCRT-II due to bi-allelic SNF8 variants is associated with a spectrum of neurodevelopmental/neurodegenerative phenotypes mediated likely via impairment of the autophagic flux.

Genetic variants affecting NQO1 protein levels impact the efficacy of idebenone treatment in Leber hereditary optic neuropathy.

Idebenone, the only approved treatment for Leber hereditary optic neuropathy (LHON), promotes recovery of visual function in up to 50% of patients, but we can neither predict nor understand the non-responders. Idebenone is reduced by the cytosolic NAD(P)H oxidoreductase I (NQO1) and directly shuttles electrons to respiratory complex III, bypassing complex I affected in LHON. We show here that two polymorphic variants drastically reduce NQO1 protein levels when homozygous or compound heterozygous. This hampers idebenone reduction. In its oxidized form, idebenone inhibits complex I, decreasing respiratory function in cells. By retrospectively analyzing a large cohort of idebenone-treated LHON patients, classified by their response to therapy, we show that patients with homozygous or compound heterozygous NQO1 variants have the poorest therapy response, particularly if carrying the m.3460G>A/MT-ND1 LHON mutation. These results suggest consideration of patient NQO1 genotype and mitochondrial DNA mutation in the context of idebenone therapy.

AFG3L2 and ACO2-Linked Dominant Optic Atrophy: Genotype-Phenotype Characterization Compared to OPA1 Patients.

PURPOSE: Heterozygous mutations in the AFG3L2 gene (encoding a mitochondrial protease indirectly reflecting on OPA1 cleavage) and ACO2 gene (encoding the mitochondrial enzyme aconitase) are associated with isolated forms of Dominant Optic Atrophy (DOA). We aimed at describing their neuro-ophthalmological phenotype as compared with classic OPA1-related DOA. DESIGN: Cross-sectional study. METHODS: The following neuro-ophthalmological parameters were collected: logMAR visual acuity (VA), color vision, mean deviation and foveal threshold at visual fields, average and sectorial retinal nerve fiber layer (RNFL), and ganglion cell layer (GCL) thickness on optical coherence tomography. ACO2 and AFG3L2 patients were compared with an age- and sex-matched group of OPA1 patients with a 1:2 ratio. All eyes were analyzed using a clustered Wilcoxon rank sum test with the Rosner-Glynn-Lee method. RESULTS: A total of 44 eyes from 23 ACO2 patients and 26 eyes from 13 AFG3L2 patients were compared with 143 eyes from 72 OPA1 patients. All cases presented with bilateral temporal-predominant optic atrophy with various degree of visual impairment. Comparison between AFG3L2 and OPA1 failed to reveal any significant difference. ACO2 patients compared to both AFG3L2 and OPA1 presented overall higher values of nasal RNFL thickness (P = .029, P = .023), average thickness (P = .012, P = .0007), and sectorial GCL thickness. These results were confirmed also comparing separately affected and subclinical patients. CONCLUSIONS: Clinically, DOA remains a fairly homogeneous entity despite the growing genetic heterogeneity. ACO2 seems to be associated with an overall better preservation of retinal ganglion cells, probably depending on the different pathogenic mechanism involving mtDNA maintenance, as opposed to AFG3L2, which is involved in OPA1 processing and is virtually indistinguishable from classic OPA1-DOA.

Recessive MECR pathogenic variants cause an LHON-like optic neuropathy.

BACKGROUND: Leber's hereditary optic neuropathy (LHON) is a mitochondrial disorder characterised by complex I defect leading to sudden degeneration of retinal ganglion cells. Although typically associated with pathogenic variants in mitochondrial DNA, LHON was recently described in patients carrying biallelic variants in nuclear genes DNAJC30, NDUFS2 and MCAT. MCAT is part of mitochondrial fatty acid synthesis (mtFAS), as also MECR, the mitochondrial trans-2-enoyl-CoA reductase. MECR mutations lead to a recessive childhood-onset syndromic disorder with dystonia, optic atrophy and basal ganglia abnormalities. METHODS: We studied through whole exome sequencing two sisters affected by sudden and painless visual loss at young age, with partial recovery and persistent central scotoma. We modelled the candidate variant in yeast and studied mitochondrial dysfunction in yeast and fibroblasts. We tested protein lipoylation and cell response to oxidative stress in yeast. RESULTS: Both sisters carried a homozygous pathogenic variant in MECR (p.Arg258Trp). In yeast, the MECR-R258W mutant showed an impaired oxidative growth, 30% reduction in oxygen consumption rate and 80% decrease in protein levels, pointing to structure destabilisation. Fibroblasts confirmed the reduced amount of MECR protein, but failed to reproduce the OXPHOS defect. Respiratory complexes assembly was normal. Finally, the yeast mutant lacked lipoylation of key metabolic enzymes and was more sensitive to H2O2 treatment. Lipoic Acid supplementation partially rescued the growth defect. CONCLUSION: We report the first family with homozygous MECR variant causing an LHON-like optic neuropathy, which pairs the recent MCAT findings, reinforcing the impairment of mtFAS as novel pathogenic mechanism in LHON.

Multimodal investigation of melanopsin retinal ganglion cells in Alzheimer's disease.

OBJECTIVE: In Alzheimer's disease (AD), the presence of circadian dysfunction is well-known and may occur early in the disease course. The melanopsin retinal ganglion cell (mRGC) system may play a relevant role in contributing to circadian dysfunction. In this study, we aimed at evaluating, through a multimodal approach, the mRGC system in AD at an early stage of disease. METHODS: We included 29 mild-moderate AD (70.9 ± 11 years) and 26 (70.5 ± 8 years) control subjects. We performed an extensive neurophtalmological evaluation including optical coherence tomography with ganglion cell layer segmentation, actigraphic evaluation of the rest-activity rhythm, chromatic pupillometry analyzed with a new data-fitting approach, and brain functional MRI combined with light stimuli assessing the mRGC system. RESULTS: We demonstrated a significant thinning of the infero-temporal sector of the ganglion cell layer in AD compared to controls. Moreover, we documented by actigraphy the presence of a circadian-impaired AD subgroup. Overall, circadian measurements worsened by age. Chromatic pupillometry evaluation highlighted the presence of a pupil-light response reduction in the rod condition pointing to mRGC dendropathy. Finally, brain fMRI showed a reduced occipital cortex activation with blue light particularly for the sustained responses. INTERPRETATION: Overall, the results of this multimodal innovative approach clearly document a dysfunctional mRGC system at early stages of disease as a relevant contributing factor for circadian impairment in AD providing also support to the use of light therapy in AD.

The Acute Optic Neuritis Network (ACON): Study protocol of a non-interventional prospective multicenter study on diagnosis and treatment of acute optic neuritis.

Optic neuritis (ON) often occurs at the presentation of multiple sclerosis (MS), neuromyelitis optica spectrum disorders (NMOSD), and myelin oligodendrocyte glycoprotein (MOG) antibody-associated disease (MOGAD). The recommended treatment of high-dose corticosteroids for ON is based on a North American study population, which did not address treatment timing or antibody serostatus. The Acute Optic Neuritis Network (ACON) presents a global, prospective, observational study protocol primarily designed to investigate the effect of time to high-dose corticosteroid treatment on 6-month visual outcomes in ON. Patients presenting within 30 days of the inaugural ON will be enrolled. For the primary analysis, patients will subsequently be assigned into the MS-ON group, the aquapotin-4-IgG positive ON (AQP4-IgG+ON) group or the MOG-IgG positive ON (MOG-IgG+ON) group and then further sub-stratified according to the number of days from the onset of visual loss to high-dose corticosteroids (days-to-Rx). The primary outcome measure will be high-contrast best-corrected visual acuity (HC-BCVA) at 6 months. In addition, multimodal data will be collected in subjects with any ON (CIS-ON, MS-ON, AQP4-IgG+ON or MOG-IgG+ON, and seronegative non-MS-ON), excluding infectious and granulomatous ON. Secondary outcomes include low-contrast best-corrected visual acuity (LC-BCVA), optical coherence tomography (OCT), magnetic resonance imaging (MRI) measurements, serum and cerebrospinal fluid (CSF) biomarkers (AQP4-IgG and MOG-IgG levels, neurofilament, and glial fibrillary protein), and patient reported outcome measures (headache, visual function in daily routine, depression, and quality of life questionnaires) at presentation at 6-month and 12-month follow-up visits. Data will be collected from 28 academic hospitals from Africa, Asia, the Middle East, Europe, North America, South America, and Australia. Planned recruitment consists of 100 MS-ON, 50 AQP4-IgG+ON, and 50 MOG-IgG+ON. This prospective, multimodal data collection will assess the potential value of early high-dose corticosteroid treatment, investigate the interrelations between functional impairments and structural changes, and evaluate the diagnostic yield of laboratory biomarkers. This analysis has the ability to substantially improve treatment strategies and the accuracy of diagnostic stratification in acute demyelinating ON. Trial registration: ClinicalTrials.gov, identifier: NCT05605951.

Mitochondrial optic neuropathies.

Mitochondrial optic neuropathies have a leading role in the field of mitochondrial medicine ever since 1988, when the first mutation in mitochondrial DNA was associated with Leber's hereditary optic neuropathy (LHON). Autosomal dominant optic atrophy (DOA) was subsequently associated in 2000 with mutations in the nuclear DNA affecting the OPA1 gene. LHON and DOA are both characterized by selective neurodegeneration of retinal ganglion cells (RGCs) triggered by mitochondrial dysfunction. This is centered on respiratory complex I impairment in LHON and defective mitochondrial dynamics in OPA1-related DOA, leading to distinct clinical phenotypes. LHON is a subacute, rapid, severe loss of central vision involving both eyes within weeks or months, with age of onset between 15 and 35 years old. DOA is a more slowly progressive optic neuropathy, usually apparent in early childhood. LHON is characterized by marked incomplete penetrance and a clear male predilection. The introduction of next-generation sequencing has greatly expanded the genetic causes for other rare forms of mitochondrial optic neuropathies, including recessive and X-linked, further emphasizing the exquisite sensitivity of RGCs to compromised mitochondrial function. All forms of mitochondrial optic neuropathies, including LHON and DOA, can manifest either as pure optic atrophy or as a more severe multisystemic syndrome. Mitochondrial optic neuropathies are currently at the forefront of a number of therapeutic programs, including gene therapy, with idebenone being the only approved drug for a mitochondrial disorder.

Indirect Comparison of Lenadogene Nolparvovec Gene Therapy Versus Natural History in Patients with Leber Hereditary Optic Neuropathy Carrying the m.11778G>A MT-ND4 Mutation.

INTRODUCTION: Lenadogene nolparvovec is a promising novel gene therapy for patients with Leber hereditary optic neuropathy (LHON) carrying the m.11778G>A ND4 mutation (MT-ND4). A previous pooled analysis of phase 3 studies showed an improvement in visual acuity of patients injected with lenadogene nolparvovec compared to natural history. Here, we report updated results by incorporating data from the latest phase 3 trial REFLECT in the pool, increasing the number of treated patients from 76 to 174. METHODS: The visual acuity of 174 MT-ND4-carrying patients with LHON injected in one or both eyes with lenadogene nolparvovec from four pooled phase 3 studies (REVERSE, RESCUE and their long-term extension trial RESTORE; and REFLECT trial) was compared to the spontaneous evolution of an external control group of 208 matched patients from 11 natural history studies. RESULTS: Treated patients showed a clinically relevant and sustained improvement in their visual acuity when compared to natural history. Mean improvement versus natural history was - 0.30 logMAR (+ 15 ETDRS letters equivalent) at last observation (P < 0.01) with a maximal follow-up of 3.9 years after injection. Most treated eyes were on-chart as compared to less than half of natural history eyes at 48 months after vision loss (89.6% versus 48.1%; P < 0.01) and at last observation (76.1% versus 44.4%; P < 0.01). When we adjusted for covariates of interest (gender, age of onset, ethnicity, and duration of follow-up), the estimated mean gain was - 0.43 logMAR (+ 21.5 ETDRS letters equivalent) versus natural history at last observation (P < 0.0001). Treatment effect was consistent across all phase 3 clinical trials. Analyses from REFLECT suggest a larger treatment effect in patients receiving bilateral injection compared to unilateral injection. CONCLUSION: The efficacy of lenadogene nolparvovec in improving visual acuity in MT-ND4 LHON was confirmed in a large cohort of patients, compared to the spontaneous natural history decline. Bilateral injection of gene therapy may offer added benefits over unilateral injection. TRIAL REGISTRATION NUMBERS: NCT02652780 (REVERSE); NCT02652767 (RESCUE); NCT03406104 (RESTORE); NCT03293524 (REFLECT); NCT03295071 (REALITY).

Childhood-Onset Leber Hereditary Optic Neuropathy-Clinical and Prognostic Insights.

PURPOSE: To investigate the clinical and molecular genetic features of childhood-onset Leber hereditary optic neuropathy (LHON) to gain a better understanding of the factors influencing the visual outcome in this atypical form of the disease. DESIGN: Retrospective cohort study. METHODS: We retrospectively included 2 cohorts of patients with LHON with onset of visual loss before the age of 12 years from Italy and the United Kingdom. Ophthalmologic evaluation, including best-corrected visual acuity, orthoptic evaluation, slit-lamp biomicroscopy, visual field testing, and optical coherence tomography, was considered. Patients were classified based on both the age of onset and the pattern of visual loss. RESULTS: A total of 68 patients were stratified based on the age of onset of visual loss: group 1 (<3 years): 14 patients (20.6%); group 2 (≥3 to <9 years): 27 patients (39.7%); and group 3 (≥9 to ≤12 years): 27 patients (39.7%). Patients in group 2 achieved a better visual outcome than those in group 3. Patients in groups 1 and 2 had better mean deviation on visual field testing than those in group 3. The mean ganglion cell layer thickness on optical coherence tomography in group 2 was higher than those in groups 1 and 3. Patients were also categorized based on the pattern of visual loss as follows: Subacute Bilateral: 54 patients (66.7%); Insidious Bilateral: 14 patients (17.3%); Unilateral: 9 patients (11.1%); and Subclinical Bilateral: 4 patients (4.9%). CONCLUSIONS: Children who lose vision from LHON before the age of 9 years have a better visual prognosis than those who become affected in later years, likely representing a "form frustre" of the disease.

Chromatic pupillometry for evaluating melanopsin retinal ganglion cell function in Alzheimer's disease and other neurodegenerative disorders: a review.

The evaluation of pupillary light reflex (PLR) by chromatic pupillometry may provide a unique insight into specific photoreceptor functions. Chromatic pupillometry refers to evaluating PLR to different wavelengths and intensities of light in order to differentiate outer/inner retinal photoreceptor contributions to the PLR. Different protocols have been tested and are now established to assess in-vivo PLR contribution mediated by melanopsin retinal ganglion cells (mRGCs). These intrinsically photosensitive photoreceptors modulate the non-image-forming functions of the eye, which are mainly the circadian photoentrainment and PLR, via projections to the hypothalamic suprachiasmatic and olivary pretectal nucleus, respectively. In this context, chromatic pupillometry has been used as an alternative and non-invasive tool to evaluate the mRGC system in several clinical settings, including hereditary optic neuropathies, glaucoma, and neurodegenerative disorders such as Parkinson's disease (PD), idiopathic/isolated rapid eye movement sleep behavior disorder (iRBD), and Alzheimer's disease (AD). The purpose of this article is to review the key steps of chromatic pupillometry protocols for studying in-vivo mRGC-system functionality and provide the main findings of this technique in the research setting on neurodegeneration. mRGC-dependent pupillary responses are short-wavelength sensitive, have a higher threshold of activation, and are much slower and sustained compared with rod- and cone-mediated responses, driving the tonic component of the PLR during exposure to high-irradiance and continuous light stimulus. Thus, mRGCs contribute mainly to the tonic component of the post-illumination pupil response (PIPR) to bright blue light flash that persists after light stimulation is switched off. Given the role of mRGCs in circadian photoentrainment, the use of chromatic pupillometry to perform a functional evaluation of mRGcs may be proposed as an early biomarker of mRGC-dysfunction in neurodegenerative disorders characterized by circadian and/or sleep dysfunction such as AD, PD, and its prodromal phase iRBD. The evaluation by chromatic pupillometry of mRGC-system functionality may lay the groundwork for a new, easily accessible biomarker that can be exploited also as the starting point for future longitudinal cohort studies aimed at stratifying the risk of conversion in these disorders.

Mammalian RNase H1 directs RNA primer formation for mtDNA replication initiation and is also necessary for mtDNA replication completion.

The in vivo role for RNase H1 in mammalian mitochondria has been much debated. Loss of RNase H1 is embryonic lethal and to further study its role in mtDNA expression we characterized a conditional knockout of Rnaseh1 in mouse heart. We report that RNase H1 is essential for processing of RNA primers to allow site-specific initiation of mtDNA replication. Without RNase H1, the RNA:DNA hybrids at the replication origins are not processed and mtDNA replication is initiated at non-canonical sites and becomes impaired. Importantly, RNase H1 is also needed for replication completion and in its absence linear deleted mtDNA molecules extending between the two origins of mtDNA replication are formed accompanied by mtDNA depletion. The steady-state levels of mitochondrial transcripts follow the levels of mtDNA, and RNA processing is not altered in the absence of RNase H1. Finally, we report the first patient with a homozygous pathogenic mutation in the hybrid-binding domain of RNase H1 causing impaired mtDNA replication. In contrast to catalytically inactive variants of RNase H1, this mutant version has enhanced enzyme activity but shows impaired primer formation. This finding shows that the RNase H1 activity must be strictly controlled to allow proper regulation of mtDNA replication.

A Second Case With the V374A KCND3 Pathogenic Variant in an Italian Patient With Early-Onset Spinocerebellar Ataxia.

Background and Objectives: To date, approximately 20 heterozygous mainly loss-of-function variants in KCND3 have been associated with spinocerebellar ataxia (SCA) type 19 and 22, a clinically heterogeneous group of neurodegenerative disorders. We aimed at reporting the second patients with the V374A KCND3 mutation from an independent family, confirming its pathogenic role. Methods: We describe the clinical history of a patient with SCA and conducted genetic investigations including mitochondrial DNA analysis and exome sequencing. Results: This male patient was reported to have unstable gait with tremors at the lower limbs and dysarthric speech since childhood. A neurologic examination also showed dysarthria, nystagmus, action tremor, dysmetria, and weak deep tendon reflexes. He had marked cerebellar atrophy at brain MRI, more evident at vermis. Molecular analysis, including exome sequencing and an in silico panel analysis of genes associated with SCA, revealed the c.1121T>C [p.V374A] mutation in KCND3. Discussion: This report consolidates the pathogenicity of the V374A KCND3 mutation and suggests that the ataxic paroxysmal exacerbations are not a key phenotypic feature of this mutation.

Rapamycin rescues mitochondrial dysfunction in cells carrying the m.8344A > G mutation in the mitochondrial tRNALys.

BACKGROUND: Myoclonus, Epilepsy and Ragged-Red-Fibers (MERRF) is a mitochondrial encephalomyopathy due to heteroplasmic mutations in mitochondrial DNA (mtDNA) most frequently affecting the tRNALys gene at position m.8344A > G. Defective tRNALys severely impairs mitochondrial protein synthesis and respiratory chain when a high percentage of mutant heteroplasmy crosses the threshold for full-blown clinical phenotype. Therapy is currently limited to symptomatic management of myoclonic epilepsy, and supportive measures to counteract muscle weakness with co-factors/supplements. METHODS: We tested two therapeutic strategies to rescue mitochondrial function in cybrids and fibroblasts carrying different loads of the m.8344A > G mutation. The first strategy was aimed at inducing mitochondrial biogenesis directly, over-expressing the master regulator PGC-1α, or indirectly, through the treatment with nicotinic acid, a NAD+ precursor. The second was aimed at stimulating the removal of damaged mitochondria through prolonged rapamycin treatment. RESULTS: The first approach slightly increased mitochondrial protein expression and respiration in the wild type and intermediate-mutation load cells, but was ineffective in high-mutation load cell lines. This suggests that induction of mitochondrial biogenesis may not be sufficient to rescue mitochondrial dysfunction in MERRF cells with high-mutation load. The second approach, when administered chronically (4 weeks), induced a slight increase of mitochondrial respiration in fibroblasts with high-mutation load, and a significant improvement in fibroblasts with intermediate-mutation load, rescuing completely the bioenergetics defect. This effect was mediated by increased mitochondrial biogenesis, possibly related to the rapamycin-induced inhibition of the Mechanistic Target of Rapamycin Complex 1 (mTORC1) and the consequent activation of the Transcription Factor EB (TFEB). CONCLUSIONS: Overall, our results point to rapamycin-based therapy as a promising therapeutic option for MERRF.