OPA1 mutation affects autophagy and triggers senescence in autosomal dominant optic atrophy plus fibroblasts.

In several cases of mitochondrial diseases, the underlying genetic and bioenergetic causes of reduced oxidative phosphorylation (OxPhos) in mitochondrial dysfunction are well understood. However, there is still limited knowledge about the specific cellular outcomes and factors involved for each gene and mutation, which contributes to the lack of effective treatments for these disorders. This study focused on fibroblasts from a patient with Autosomal Dominant Optic Atrophy (ADOA) plus syndrome harboring a mutation in the Optic Atrophy 1 (OPA1) gene. By combining functional and transcriptomic approaches, we investigated the mitochondrial function and identified cellular phenotypes associated with the disease. Our findings revealed that fibroblasts with the OPA1 mutation exhibited a disrupted mitochondrial network and function, leading to altered mitochondrial dynamics and reduced autophagic response. Additionally, we observed a premature senescence phenotype in these cells, suggesting a previously unexplored role of the OPA1 gene in inducing senescence in ADOA plus patients. This study provides novel insights into the mechanisms underlying mitochondrial dysfunction in ADOA plus and highlights the potential importance of senescence in disease progression.

Extreme phenotypic heterogeneity in non-expansion spinocerebellar ataxias.

Although the best-known spinocerebellar ataxias (SCAs) are triplet repeat diseases, many SCAs are not caused by repeat expansions. The rarity of individual non-expansion SCAs, however, has made it difficult to discern genotype-phenotype correlations. We therefore screened individuals who had been found to bear variants in a non-expansion SCA-associated gene through genetic testing, and after we eliminated genetic groups that had fewer than 30 subjects, there were 756 subjects bearing single-nucleotide variants or deletions in one of seven genes: CACNA1A (239 subjects), PRKCG (175), AFG3L2 (101), ITPR1 (91), STUB1 (77), SPTBN2 (39), or KCNC3 (34). We compared age at onset, disease features, and progression by gene and variant. There were no features that reliably distinguished one of these SCAs from another, and several genes-CACNA1A, ITPR1, SPTBN2, and KCNC3-were associated with both adult-onset and infantile-onset forms of disease, which also differed in presentation. Nevertheless, progression was overall very slow, and STUB1-associated disease was the fastest. Several variants in CACNA1A showed particularly wide ranges in age at onset: one variant produced anything from infantile developmental delay to ataxia onset at 64 years of age within the same family. For CACNA1A, ITPR1, and SPTBN2, the type of variant and charge change on the protein greatly affected the phenotype, defying pathogenicity prediction algorithms. Even with next-generation sequencing, accurate diagnosis requires dialogue between the clinician and the geneticist.

Mitofusin 2 mutation drives cell proliferation in Charcot-Marie-Tooth 2A fibroblasts.

Dominant mutations in ubiquitously expressed mitofusin 2 gene (MFN2) cause Charcot-Marie-Tooth type 2A (CMT2A; OMIM 609260), an inherited sensory-motor neuropathy that affects peripheral nerve axons. Mitofusin 2 protein has been found to take part in mitochondrial fusion, mitochondria-endoplasmic reticulum tethering, mitochondrial trafficking along axons, mitochondrial quality control and various types of cancer, in which MFN2 has been indicated as a tumor suppressor gene. Discordant data on the mitochondrial altered phenotypes in patient-derived fibroblasts harboring MFN2 mutations and in animal models have been reported. We addressed some of these issues by focusing on mitochondria behavior during autophagy and mitophagy in fibroblasts derived from a CMT2AMFN2 patient with an MFN2650G > T/C217F mutation in the GTPase domain. This study investigated mitochondrial dynamics, respiratory capacity and autophagy/mitophagy, to tackle the multifaceted MFN2 contribution to CMT2A pathogenesis. We found that MFN2 mutated fibroblasts showed impairment of mitochondrial morphology, bioenergetics capacity, and impairment of the early stages of autophagy, but not mitophagy. Unexpectedly, transcriptomic analysis of mutated fibroblasts highlighted marked differentially expressed pathways related to cell population proliferation and extracellular matrix organization. We consistently found the activation of mTORC2/AKT signaling and accelerated proliferation in the CMT2AMFN2 fibroblasts. In conclusion, our evidence indicates that MFN2 mutation can positively drive cell proliferation in CMT2AMFN2 fibroblasts.

Decreasing ganglioside synthesis delays motor and cognitive symptom onset in Spg11 knockout mice.

Biallelic variants in the SPG11 gene account for the most common form of autosomal recessive hereditary spastic paraplegia characterized by motor and cognitive impairment, with currently no therapeutic option. We previously observed in a Spg11 knockout mouse that neurodegeneration is associated with accumulation of gangliosides in lysosomes. To test whether a substrate reduction therapy could be a therapeutic option, we downregulated the key enzyme involved in ganglioside biosynthesis using an AAV-PHP.eB viral vector expressing a miRNA targeting St3gal5. Downregulation of St3gal5 in Spg11 knockout mice prevented the accumulation of gangliosides, delayed the onset of motor and cognitive symptoms, and prevented the upregulation of serum levels of neurofilament light chain, a biomarker widely used in neurodegenerative diseases. Importantly, similar results were observed when Spg11 knockout mice were administrated venglustat, a pharmacological inhibitor of glucosylceramide synthase expected to decrease ganglioside synthesis. Downregulation of St3gal5 or venglustat administration in Spg11 knockout mice strongly decreased the formation of axonal spheroids, previously associated with impaired trafficking. Venglustat had similar effect on cultured human SPG11 neurons. In conclusion, this work identifies the first disease-modifying therapeutic strategy in SPG11, and provides data supporting its relevance for therapeutic testing in SPG11 patients.

Targeting autophagy impairment improves the phenotype of a novel CLN8 zebrafish model.

CLN8 is an endoplasmic reticulum cargo receptor and a regulator of lysosome biogenesis whose loss of function leads to neuronal ceroid lipofuscinosis. CLN8 has been linked to autophagy and lipid metabolism, but much remains to be learned, and there are no therapies acting on the molecular signatures in this disorder. The present study aims to characterize the molecular pathways involved in CLN8 disease and, by pinpointing altered ones, to identify potential therapies. To bridge the gap between cell and mammalian models, we generated a new zebrafish model of CLN8 deficiency, which recapitulates the pathological features of the disease. We observed, for the first time, that CLN8 dysfunction impairs autophagy. Using autophagy modulators, we showed that trehalose and SG2 are able to attenuate the pathological phenotype in mutant larvae, confirming autophagy impairment as a secondary event in disease progression. Overall, our successful modeling of CLN8 defects in zebrafish highlights this novel in vivo model's strong potential as an instrument for exploring the role of CLN8 dysfunction in cellular pathways, with a view to identifying small molecules to treat this rare disease.

Responsiveness of the Scale for the Assessment and Rating of Ataxia and Natural History in 884 Recessive and Early Onset Ataxia Patients.

OBJECTIVE: The Scale for the Assessment and Rating of Ataxia (SARA) is the most widely applied clinical outcome assessment (COA) for genetic ataxias, but presents metrological and regulatory challenges. To facilitate trial planning, we characterize its responsiveness (including subitem-level relations to ataxia severity and patient-focused outcomes) across a large number of ataxias, and provide first natural history data for several of them. METHODS: Subitem-level correlation and distribution-based analysis of 1,637 SARA assessments in 884 patients with autosomal recessive/early onset ataxia (370 with 2-8 longitudinal assessments) were complemented by linear mixed effects modeling to estimate progression and sample sizes. RESULTS: Although SARA subitem responsiveness varied between ataxia severities, gait/stance showed a robust granular linear scaling across the broadest range (SARA < 25). Responsiveness was diminished by incomplete subscale use at intermediate or upper levels, nontransitions ("static periods"), and fluctuating decreases/increases. All subitems except nose-finger showed moderate-to-strong correlations to activities of daily living, indicating that metric properties-not content validity-limit SARA responsiveness. SARA captured mild-to-moderate progression in many genotypes (eg, SYNE1-ataxia: 0.55 points/yr, ataxia with oculomotor apraxia type 2: 1.14 points/yr, POLG-ataxia: 1.56 points/yr), but no change in others (autosomal recessive spastic ataxia of Charlevoix-Saguenay, COQ8A-ataxia). Whereas sensitivity to change was optimal in mild ataxia (SARA < 10), it substantially deteriorated in advanced ataxia (SARA > 25; 2.7-fold sample size). Use of a novel rank-optimized SARA without subitems finger-chase and nose-finger reduces sample sizes by 20 to 25%. INTERPRETATION: This study comprehensively characterizes COA properties and annualized changes of the SARA across and within a large number of ataxias. It suggests specific approaches for optimizing its responsiveness that might facilitate regulatory qualification and trial design. ANN NEUROL 2023;94:470-485.

Digital Gait Outcomes for Autosomal Recessive Spastic Ataxia of Charlevoix-Saguenay (ARSACS): Discriminative, Convergent, and Ecological Validity in a Multicenter Study (PROSPAX).

BACKGROUND: With treatment trials on the horizon, this study aimed to identify candidate digital-motor gait outcomes for autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS), capturable by wearable sensors with multicenter validity, and ideally also ecological validity during free walking outside laboratory settings. METHODS: Cross-sectional multicenter study (four centers), with gait assessments in 36 subjects (18 ARSACS patients; 18 controls) using three body-worn sensors (Opal, APDM) in laboratory settings and free walking in public spaces. Sensor gait measures were analyzed for discriminative validity from controls, and for convergent (ie, clinical and patient relevance) validity by correlations with SPRSmobility (primary outcome) and Scale for the Assessment and Rating of Ataxia (SARA), Spastic Paraplegia Rating Scale (SPRS), and activities of daily living subscore of the Friedreich Ataxia Rating Scale (FARS-ADL) (exploratory outcomes). RESULTS: Of 30 hypothesis-based digital gait measures, 14 measures discriminated ARSACS patients from controls with large effect sizes (|Cliff's δ| > 0.8) in laboratory settings, with strongest discrimination by measures of spatiotemporal variability Lateral Step Deviation (δ = 0.98), SPcmp (δ = 0.94), and Swing CV (δ = 0.93). Large correlations with the SPRSmobility were observed for Swing CV (Spearman's ρ = 0.84), Speed (ρ = -0.63), and Harmonic Ratio V (ρ = -0.62). During supervised free walking in a public space, 11/30 gait measures discriminated ARSACS from controls with large effect sizes. Large correlations with SPRSmobility were here observed for Swing CV (ρ = 0.78) and Speed (ρ = -0.69), without reductions in effect sizes compared with laboratory settings. CONCLUSIONS: We identified a promising set of digital-motor candidate gait outcomes for ARSACS, applicable in multicenter settings, correlating with patient-relevant health aspects, and with high validity also outside laboratory settings, thus simulating real-life walking with higher ecological validity. © 2024 The Author(s). Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

FARS-ADL across Ataxias: Construct Validity, Sensitivity to Change, and Minimal Important Change.

BACKGROUND: Patient-focused outcomes present a central need for trial-readiness across all ataxias. The Activities of Daily Living part of the Friedreich Ataxia Rating Scale (FARS-ADL) captures functional impairment and longitudinal change but is only validated in Friedreich Ataxia. OBJECTIVE: Validation of FARS-ADL regarding disease severity and patient-meaningful impairment, and its sensitivity to change across genetic ataxias. METHODS: Real-world registry data of FARS-ADL in 298 ataxia patients across genotypes were analyzed, including (1) cross-correlation with FARS-stage, Scale for the Assessment and Rating of Ataxia (SARA), Patient-Reported Outcome Measure (PROM)-ataxia, and European Quality of Life 5 Dimensions visual analogue scale (EQ5D-VAS); (2) sensitivity to change within a trial-relevant 1-year median follow-up, anchored in Patient Global Impression of Change (PGI-C); and (3) general linear modeling of factors age, sex, and depression (nine-item Patient Health Questionnaire [PHQ-9]). RESULTS: FARS-ADL correlated with overall disability (rhoFARS-stage = 0.79), clinical disease severity (rhoSARA = 0.80), and patient-reported impairment (rhoPROM-ataxia = 0.69, rhoEQ5D-VAS = -0.37), indicating comprehensive construct validity. Also at item level, and validated within genotype (SCA3, RFC1), FARS-ADL correlated with the corresponding SARA effector domains; and all items correlated to EQ5D-VAS quality of life. FARS-ADL was sensitive to change at a 1-year interval, progressing only in patients with worsening PGI-C. Minimal important change was 1.1. points based on intraindividual variability in patients with stable PGI-C. Depression was captured using FARS-ADL (+0.3 points/PHQ-9 count) and EQ5D-VAS, but not FARS-stage or SARA. CONCLUSION: FARS-ADL reflects both disease severity and patient-meaningful impairment across genetic ataxias, with sensitivity to change in trial-relevant timescales in patients perceiving change. It thus presents a promising patient-focused outcome for upcoming ataxia trials. © 2024 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society.

The clinical and molecular spectrum of ZFYVE26-associated hereditary spastic paraplegia: SPG15.

In the field of hereditary spastic paraplegia (HSP), progress in molecular diagnostics needs to be translated into robust phenotyping studies to understand genetic and phenotypic heterogeneity and to support interventional trials. ZFYVE26-associated hereditary spastic paraplegia (HSP-ZFYVE26, SPG15) is a rare, early-onset complex HSP, characterized by progressive spasticity and a variety of other neurological symptoms. While prior reports, often in populations with high rates of consanguinity, have established a general phenotype, there is a lack of systematic investigations and a limited understanding of age-dependent manifestation of symptoms. Here we delineate the clinical, neuroimaging and molecular features of 44 individuals from 36 families, the largest cohort assembled to date. Median age at last follow-up was 23.8 years covering a wide age range (11-61 years). While symptom onset often occurred in early childhood [median: 24 months, interquartile range (IQR) = 24], a molecular diagnosis was reached at a median age of 18.8 years (IQR = 8), indicating significant diagnostic delay. We demonstrate that most patients present with motor and/or speech delay or learning disabilities. Importantly, these developmental symptoms preceded the onset of motor symptoms by several years. Progressive spasticity in the lower extremities, the hallmark feature of HSP-ZFYVE26, typically presents in adolescence and involves the distal lower limbs before progressing proximally. Spasticity in the upper extremities was seen in 64%. We found a high prevalence of extrapyramidal movement disorders including cerebellar ataxia (64%) and dystonia (11%). Parkinsonism (16%) was present in a subset and showed no sustained response to levodopa. Cognitive decline and neurogenic bladder dysfunction progressed over time in most patients. A systematic analysis of brain MRI features revealed a common diagnostic signature consisting of thinning of the anterior corpus callosum, signal changes of the anterior forceps and non-specific cortical and cerebellar atrophy. The molecular spectrum included 45 distinct variants, distributed across the protein structure without mutational hotspots. Spastic Paraplegia Rating Scale scores, SPATAX Disability Scores and the Four Stage Functional Mobility Score showed moderate strength in representing the proportion of variation between disease duration and motor dysfunction. Plasma neurofilament light chain levels were significantly elevated in all patients (Mann-Whitney U-test, P < 0.0001) and were correlated inversely with age (Spearman's rank correlation coefficient r = -0.65, P = 0.01). In summary, our systematic cross-sectional analysis of HSP-ZFYVE26 patients across a wide age-range, delineates core clinical, neuroimaging and molecular features and identifies markers of disease severity. These results raise awareness to this rare disease, facilitate an early diagnosis and create clinical trial readiness.

Power of NGS-based tests in HSP diagnosis: analysis of massively parallel sequencing in clinical practice.

Hereditary spastic paraplegia (HSP) refers to a group of heterogeneous neurological disorders mainly characterized by corticospinal degeneration (pure forms), but sometimes associated with additional neurological and extrapyramidal features (complex HSP). The advent of next-generation sequencing (NGS) has led to huge improvements in knowledge of HSP genetics and made it possible to clarify the genetic etiology of hundreds of "cold cases," accelerating the process of reaching a molecular diagnosis. The different NGS-based strategies currently employed as first-tier approaches most commonly involve the use of targeted resequencing panels and exome sequencing, whereas genome sequencing remains a second-tier approach because of its high costs. The question of which approach is the best is still widely debated, and many factors affect the choice. Here, we aim to analyze the diagnostic power of different NGS techniques applied in HSP, by reviewing 38 selected studies in which different strategies were applied in different-sized cohorts of patients with genetically uncharacterized HSP.

Plasma Neurofilament Light Chain Is Elevated in Adaptor Protein Complex 4-Related Hereditary Spastic Paraplegia.

BACKGROUND: Adaptor protein complex 4-associated hereditary spastic paraplegia (AP-4-HSP) is caused by pathogenic biallelic variants in AP4B1, AP4M1, AP4E1, and AP4S1. OBJECTIVE: The aim was to explore blood markers of neuroaxonal damage in AP-4-HSP. METHODS: Plasma neurofilament light chain (pNfL) and glial fibrillary acidic protein (GFAP) levels were measured in samples from patients and age- and sex-matched controls (NfL: n = 46 vs. n = 46; GFAP: n = 14 vs. n = 21) using single-molecule array assays. Patients' phenotypes were systematically assessed using the AP-4-HSP natural history study questionnaires, the Spastic Paraplegia Rating Scale, and the SPATAX disability score. RESULTS: pNfL levels increased in AP-4-HSP patients, allowing differentiation from controls (Mann-Whitney U test: P = 3.0e-10; area under the curve = 0.87 with a 95% confidence interval of 0.80-0.94). Phenotypic cluster analyses revealed a subgroup of individuals with severe generalized-onset seizures and developmental stagnation, who showed differentially higher pNfL levels (Mann-Whitney U test between two identified clusters: P = 2.5e-6). Plasma GFAP levels were unchanged in patients with AP-4-HSP. CONCLUSIONS: pNfL is a potential disease marker in AP-4-HSP and can help differentiate between phenotypic subgroups. © 2023 International Parkinson and Movement Disorder Society.

Standards of Fluid Biomarker Collection and Pre-analytical Processes in Humans and Mice: Recommendations by the Ataxia Global Initiative Working Group on Biomarkers.

The Ataxia Global Initiative (AGI) aims to serve as a platform to facilitate clinical trial readiness for the hereditary ataxias. Clinical trials for these diseases have been hampered by the lack of objective measures to study disease onset, progression, and treatment efficacy. While these issues are not unique to the genetic ataxias, the relative rarity of these diseases makes the need for such measures even more pressing to achieve statistical power in clinical trials. In this report, we have described the efforts of the AGI fluid biomarker working group (WG) in developing uniform protocols for biomarker sampling and storage, both for human and preclinical studies in mice. By reducing collection variability, we anticipate reduced noise in downstream biomarker analysis that will improve statistical power and minimize the necessary sample size. The emphasis has been on defining and standardizing the sampling and pre-analytical work-up of minimal set of biological samples, specifically blood plasma and serum, keeping in mind the need for harmonization of collection and storage that can be achieved with relatively limited cost and resources. An optional package is detailed for those centers that have the resources and commitment for additional biofluids/sample processing and storage. Finally, we have delineated similar standardized protocols for mice that will be important for preclinical studies in the field.

New cellular imaging-based method to distinguish the SPG4 subtype of hereditary spastic paraplegia.

BACKGROUND AND PURPOSE: Microtubule defects are a common feature in several neurodegenerative disorders, including hereditary spastic paraplegia. The most frequent form of hereditary spastic paraplegia is caused by mutations in the SPG4/SPAST gene, encoding the microtubule severing enzyme spastin. To date, there is no effective therapy available but spastin-enhancing therapeutic approaches are emerging; thus prognostic and predictive biomarkers are urgently required. METHODS: An automated, simple, fast and non-invasive cell imaging-based method was developed to quantify microtubule cytoskeleton organization changes in lymphoblastoid cells and peripheral blood mononuclear cells. RESULTS: It was observed that lymphoblastoid cells and peripheral blood mononuclear cells from individuals affected by SPG4-hereditary spastic paraplegia show a polarized microtubule cytoskeleton organization. In a pilot study on freshly isolated peripheral blood mononuclear cells, our method discriminates SPG4-hereditary spastic paraplegia from healthy donors and other hereditary spastic paraplegia subtypes. In addition, it is shown that our method can detect the effects of spastin protein level changes. CONCLUSIONS: These findings open the possibility of a rapid, non-invasive, inexpensive test useful to recognize SPG4-hereditary spastic paraplegia subtype and evaluate the effects of spastin-enhancing drug in non-neuronal cells.

Converging Role for REEP1/SPG31 in Oxidative Stress.

Mutations in the receptor expression-enhancing protein 1 gene (REEP1) are associated with hereditary spastic paraplegia type 31 (SPG31), a neurological disorder characterized by length-dependent degeneration of upper motor neuron axons. Mitochondrial dysfunctions have been observed in patients harboring pathogenic variants in REEP1, suggesting a key role of bioenergetics in disease-related manifestations. Nevertheless, the regulation of mitochondrial function in SPG31 remains unclear. To elucidate the pathophysiology underlying REEP1 deficiency, we analyzed in vitro the impact of two different mutations on mitochondrial metabolism. Together with mitochondrial morphology abnormalities, loss-of-REEP1 expression highlighted a reduced ATP production with increased susceptibility to oxidative stress. Furthermore, to translate these findings from in vitro to preclinical models, we knocked down REEP1 in zebrafish. Zebrafish larvae showed a significant defect in motor axon outgrowth leading to motor impairment, mitochondrial dysfunction, and reactive oxygen species accumulation. Protective antioxidant agents such as resveratrol rescued free radical overproduction and ameliorated the SPG31 phenotype both in vitro and in vivo. Together, our findings offer new opportunities to counteract neurodegeneration in SPG31.

Short-Term Effects of Human versus Bovine Sialylated Milk Oligosaccharide Microinjection on Zebrafish Larvae Survival, Locomotor Behavior and Gene Expression.

Milk oligosaccharides are a complex class of carbohydrates that act as bioactive factors in numerous defensive and physiological functions, including brain development. Early nutrition can modulate nervous system development and can lead to epigenetic imprinting. We attempted to increase the sialylated oligosaccharide content of zebrafish yolk reserves, with the aim of evaluating any short-term effects of the treatment on mortality, locomotor behavior, and gene expression. Wild-type embryos were microinjected with saline solution or solutions containing sialylated milk oligosaccharides extracted from human and bovine milk. The results suggest that burst activity and larval survival rates were unaffected by the treatments. Locomotion parameters were found to be similar during the light phase between control and treated larvae; in the dark, however, milk oligosaccharide-treated larvae showed increased test plate exploration. Thigmotaxis results did not reveal significant differences in either the light or the dark conditions. The RNA-seq analysis indicated that both treatments exert an antioxidant effect in developing fish. Moreover, sialylated human milk oligosaccharides seemed to increase the expression of genes related to cell cycle control and chromosomal replication, while bovine-derived oligosaccharides caused an increase in the expression of genes involved in synaptogenesis and neuronal signaling. These data shed some light on this poorly explored research field, showing that both human and bovine oligosaccharides support brain proliferation and maturation.

Adaptor protein complex 4 deficiency: a paradigm of childhood-onset hereditary spastic paraplegia caused by defective protein trafficking.

Deficiency of the adaptor protein complex 4 (AP-4) leads to childhood-onset hereditary spastic paraplegia (AP-4-HSP): SPG47 (AP4B1), SPG50 (AP4M1), SPG51 (AP4E1) and SPG52 (AP4S1). This study aims to evaluate the impact of loss-of-function variants in AP-4 subunits on intracellular protein trafficking using patient-derived cells. We investigated 15 patient-derived fibroblast lines and generated six lines of induced pluripotent stem cell (iPSC)-derived neurons covering a wide range of AP-4 variants. All patient-derived fibroblasts showed reduced levels of the AP4E1 subunit, a surrogate for levels of the AP-4 complex. The autophagy protein ATG9A accumulated in the trans-Golgi network and was depleted from peripheral compartments. Western blot analysis demonstrated a 3-5-fold increase in ATG9A expression in patient lines. ATG9A was redistributed upon re-expression of AP4B1 arguing that mistrafficking of ATG9A is AP-4-dependent. Examining the downstream effects of ATG9A mislocalization, we found that autophagic flux was intact in patient-derived fibroblasts both under nutrient-rich conditions and when autophagy is stimulated. Mitochondrial metabolism and intracellular iron content remained unchanged. In iPSC-derived cortical neurons from patients with AP4B1-associated SPG47, AP-4 subunit levels were reduced while ATG9A accumulated in the trans-Golgi network. Levels of the autophagy marker LC3-II were reduced, suggesting a neuron-specific alteration in autophagosome turnover. Neurite outgrowth and branching were reduced in AP-4-HSP neurons pointing to a role of AP-4-mediated protein trafficking in neuronal development. Collectively, our results establish ATG9A mislocalization as a key marker of AP-4 deficiency in patient-derived cells, including the first human neuron model of AP-4-HSP, which will aid diagnostic and therapeutic studies.

Bi-allelic variants in MDH2: Expanding the clinical phenotype.

Bi-allelic alterations in the MDH2 gene have recently been reported in three unrelated toddlers with early-onset severe encephalopathy. Here, we describe a new case of a child carrying novel variants in MDH2. This child presented with early-onset encephalocardiopathy requiring heart transplant and showed cerebellar ataxia and drug-responsive epilepsy; his family history was significant for multiple cancers, a feature often associated with monoallelic variants in MDH2. Functional studies in cultured skin fibroblasts from the proband showed reduced protein levels and impaired enzyme activity, further corroborating the genetic results. The relatively mild neurological presentation and severe cardiac manifestations requiring heart transplant distinguish this case from previous reports. This patient thus expands the spectrum of clinical features associated with MDH2 variants.

De Novo and Dominantly Inherited SPTAN1 Mutations Cause Spastic Paraplegia and Cerebellar Ataxia.

BACKGROUND: Pathogenic variants in SPTAN1 have been linked to a remarkably broad phenotypical spectrum. Clinical presentations include epileptic syndromes, intellectual disability, and hereditary motor neuropathy. OBJECTIVES: We investigated the role of SPTAN1 variants in rare neurological disorders such as ataxia and spastic paraplegia. METHODS: We screened 10,000 NGS datasets across two international consortia and one local database, indicative of the level of international collaboration currently required to identify genes causative for rare disease. We performed in silico modeling of the identified SPTAN1 variants. RESULTS: We describe 22 patients from 14 families with five novel SPTAN1 variants. Of six patients with cerebellar ataxia, four carry a de novo SPTAN1 variant and two show a sporadic inheritance. In this group, one variant (p.Lys2083del) is recurrent in four patients. Two patients have novel de novo missense mutations (p.Arg1098Cys, p.Arg1624Cys) associated with cerebellar ataxia, in one patient accompanied by intellectual disability and epilepsy. We furthermore report a recurrent missense mutation (p.Arg19Trp) in 15 patients with spastic paraplegia from seven families with a dominant inheritance pattern in four and a de novo origin in one case. One further patient carrying a de novo missense mutation (p.Gln2205Pro) has a complex spastic ataxic phenotype. Through protein modeling we show that mutated amino acids are located at crucial interlinking positions, interconnecting the three-helix bundle of a spectrin repeat. CONCLUSIONS: We show that SPTAN1 is a relevant candidate gene for ataxia and spastic paraplegia. We suggest that for the mutations identified in this study, disruption of the interlinking of spectrin helices could be a key feature of the pathomechanism. © 2022 International Parkinson and Movement Disorder Society.

Biallelic mutations in RNF220 cause laminopathies featuring leukodystrophy, ataxia and deafness.

Leukodystrophies are a heterogeneous group of rare inherited disorders that mostly involve the white matter of the CNS. These conditions are characterized by primary glial cell and myelin sheath pathology of variable aetiology, which causes secondary axonal degeneration, generally emerging with disease progression. Whole exome sequencing performed in five large consanguineous nuclear families allowed us to identify homozygosity for two recurrent missense variants affecting highly conserved residues of RNF220 as the causative event underlying a novel form of leukodystrophy with ataxia and sensorineural deafness. We report these two homozygous missense variants (p.R363Q and p.R365Q) in the ubiquitin E3 ligase RNF220 as the underlying cause of this novel form of leukodystrophy with ataxia and sensorineural deafness that includes fibrotic cardiomyopathy and hepatopathy as associated features in seven consanguineous families. Mass spectrometry analysis identified lamin B1 as the RNF220 binding protein and co-immunoprecipitation experiments demonstrated reduced binding of both RNF220 mutants to lamin B1. We demonstrate that RNF220 silencing in Drosophila melanogaster specifically affects proper localization of lamin Dm0, the fly lamin B1 orthologue, promotes its aggregation and causes a neurodegenerative phenotype, strongly supporting the functional link between RNF220 and lamin B1. Finally, we demonstrate that RNF220 plays a crucial role in the maintenance of nuclear morphology; mutations in primary skin fibroblasts determine nuclear abnormalities such as blebs, herniations and invaginations, which are typically observed in cells of patients affected by laminopathies. Overall, our data identify RNF220 as a gene implicated in leukodystrophy with ataxia and sensorineural deafness and document a critical role of RNF220 in the regulation of nuclear lamina. Our findings provide further evidence on the direct link between nuclear lamina dysfunction and neurodegeneration.

Biallelic variants in HPDL cause pure and complicated hereditary spastic paraplegia.

Human 4-hydroxyphenylpyruvate dioxygenase-like (HPDL) is a putative iron-containing non-heme oxygenase of unknown specificity and biological significance. We report 25 families containing 34 individuals with neurological disease associated with biallelic HPDL variants. Phenotypes ranged from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spasticity and global developmental delays, sometimes complicated by episodes of neurological and respiratory decompensation. Variants included bona fide pathogenic truncating changes, although most were missense substitutions. Functionality of variants could not be determined directly as the enzymatic specificity of HPDL is unknown; however, when HPDL missense substitutions were introduced into 4-hydroxyphenylpyruvate dioxygenase (HPPD, an HPDL orthologue), they impaired the ability of HPPD to convert 4-hydroxyphenylpyruvate into homogentisate. Moreover, three additional sets of experiments provided evidence for a role of HPDL in the nervous system and further supported its link to neurological disease: (i) HPDL was expressed in the nervous system and expression increased during neural differentiation; (ii) knockdown of zebrafish hpdl led to abnormal motor behaviour, replicating aspects of the human disease; and (iii) HPDL localized to mitochondria, consistent with mitochondrial disease that is often associated with neurological manifestations. Our findings suggest that biallelic HPDL variants cause a syndrome varying from juvenile-onset pure hereditary spastic paraplegia to infantile-onset spastic tetraplegia associated with global developmental delays.