Mitochondrial integrity is fundamental to cellular function, upheld by a network of proteases that regulate proteostasis and mitochondrial dynamics. Among these proteases, AFG3L2 is critical due to its roles in maintaining mitochondrial homeostasis, regulating mitochondrial protein quality, and facilitating mitochondrial biogenesis. Mutations in AFG3L2 are implicated in a spectrum of diseases, including spinocerebellar ataxia type 28 (SCA28) and spastic ataxia 5 (SPAX5), as well as other systemic conditions. This study employs a multi-omics approach to investigate the biochemical impact of AFG3L2 mutations in immortalized lymphoblastoid cell lines derived from a patient with biallelic variants leading to spastic ataxia (SPAX5). Our proteomic analysis revealed AFG3L2 impairment, with significant dysregulation of proteins critical for mitochondrial function, cytoskeletal integrity, and cellular metabolism. Specifically, disruptions were observed in mitochondrial dynamics and calcium homeostasis, alongside downregulation of key proteins like COX11, a copper chaperone for complex IV assembly, and NFU1, an iron-sulfur cluster protein linked to spastic paraparesis and infection-related worsening. Lipidomic analysis highlighted substantial alterations in lipid composition, with significant decreases in sphingomyelins, phosphatidylethanolamine, and phosphatidylcholine, reflecting disruptions in lipid metabolism and membrane integrity. Metabolomic profiling did not reveal any significant findings. Our comprehensive investigation into loss of functional AFG3L2 elucidates a pathophysiology extending beyond mitochondrial proteostasis, implicating a wide array of cellular processes. The findings reveal substantial cellular disturbances at multiple levels, contributing to neurodegeneration through disrupted mitochondrial respiratory chain, calcium homeostasis, cytoskeletal integrity, and altered lipid homeostasis. This study underscores the complexity of SPAX5 pathophysiology and the importance of multi-omics approaches in developing effective strategies to address the impact of loss of functional AFG3L2. Our data also highlight the value of immortalized lymphoblastoid cells as a tool for pre-clinical testing and research, offering a detailed biochemical fingerprint that enhances our understanding of SPAX5 and identifies potential areas for further investigation.

We present a case of rare autosomal dominant spinocerebellar ataxia type 28 caused by a heterozygous mutation of the AFG3L2 gene (SCA 28, SCA-AFG3L2, OMIM: 610246). The clinical presentation included a combination of visual and oculomotor disorders, extrapyramidal syndrome, epilepsy, and cerebellar ataxia. Female proband, 21 years old, 2 years of observation of the patient. Severe myopia has been reported since childhood. The disease manifested at the age of 14 with hand tremor and oculomotor disorders. Symptoms progressed over 5 years with an increase in oculomotor disorders, tremor, hypokinesia, cervical dystonia, left hand dystonia, elements of cerebellar ataxia, and the onset of epilepsy. Her parents were divorced; no data on the father was available; the family history was not reported until the relationship was restored. The proband's father and half-sister have similar symptoms, according to the proband (not medically confirmed by the authors). The patient was followed up for a long time with a diagnosis of a functional disorder of the nervous system. Type 28 spinocerebellar ataxia was diagnosed at the age of 19 years. Complete genome sequencing was performed; a heterozygous mutation c.838C >T (p.Arg280Trp) in the AFG3L2 gene was detected, and the mutation was confirmed using Sanger method. Direct Sanger sequencing was performed for the proband's father, and a similar mutation in the heterozygous state was confirmed. The proband's healthy mother had no such mutation. Segregation of these mutations in the family by the autosomal dominant type was found. The patient received levodopa 1250 mg/day, lamotrigine 400 mg/day, lacosamide 200 mg/day, as well as botulinum therapy for the muscles of the neck and left hand. Tremor and hypokinesia responded to levodopa. Sporadic epileptic seizures persist. The patient is socialized and studies at a higher educational institution. Описан случай редкой аутосомно-доминантной спиноцеребеллярной атаксии 28 типа, обусловленной гетерозиготной мутацией гена AFG3L2 (СЦА 28, SCA-AFG3L2, OMIM: 610246). Клиническая картина представлена сочетанием патологии зрения, глазодвигательных нарушений, экстрапирамидного синдрома, эпилепсии и мозжечковой атаксии. Пробанд женщина, 21 год, 2 года наблюдения за пациентом. С детства отмечалась миопия тяжелой степени. Дебют заболевания с 14 лет с развития тремора рук, глазодвигательных нарушений. Прогрессирование симптоматики в течение 5 лет с нарастанием глазодвигательных нарушений, тремора, гипокинезии, цервикальной дистонии, дистонии левой руки, элементов мозжечковой атаксии, развития эпилепсии. Родители разведены, данные об отце отсутствовали, наследственный анамнез не подтверждался до момента восстановления отношений. У отца пробанда и сводной сестры подобная симптоматика со слов пробанда (не оценивались авторами клинически). Длительное время наблюдалась с диагнозом функционального расстройства нервной системы. Диагноз «Спиноцеребеллярная атаксия 28 типа» установлен в возрасте 19 лет. Выполнено полное секвенирование генома, выявлена гетерозиготная мутация c.838C>T (p.Arg280Trp) в гене AFG3L2, мутация подтверждена по Сэнгеру. Выполнено прямое секвенирование по Сэнгеру у отца пробанда, подтверждено наличие аналогичной мутации в гетерозиготном состоянии и исключено у ее здоровой матери. Установлена сегрегация данных мутаций в семье по аутосомно-доминантному типу. Пациентка получает препараты леводопы в суточной дозе 1250 мг/сутки, ламотриджин 400 мг/сутки, а также лакосамид 200 мг/сутки, ботулинотерапию мышц шеи и левой руки. Тремор и гипокинезия были чувствительны к препаратам леводопы. Сохраняются единичные эпилептические приступы. Социализирована, обучается в высшем учебном заведении. AFG3L2-related neurologic disorders comprise four phenotypes. Spinocerebellar ataxia type 28 (SCA28), the most common phenotype, is characterized by young adult onset (26.5 ± 17.2 years); the onset range is from birth to 74 years of a cerebellar syndrome manifesting initially as very slowly progressive gait and limb ataxia resulting in incoordination and balance problems. Less frequently, ptosis/ophthalmoplegia, dysarthria, or upper-limb incoordination may occur as the initial finding. Pyramidal syndrome (increased and brisk reflexes, extensor plantar reflex, and spasticity) is commonly observed in individuals with longer disease duration. Although cognitive impairment, spasticity, and ophthalmologic signs can occur with disease progression, most individuals remain ambulatory and fully independent throughout their lives. Spastic ataxia type 5 (SPAX5), reported in 14 individuals to date, ranges from severe neurodegeneration with microcephaly, poor weight gain, developmental delay, developmental regression around age nine months, and death as early as age 2.5 years. Milder presentations range from onset in infancy to an early-onset complex cerebellar ataxia with myoclonic epilepsy. AFG3L2-related autosomal recessive spinocerebellar ataxia (AFG3L2-SCAR), reported in two individuals to date, is a late-onset ataxia with a clinical phenotype closely resembling that of SCA28. Optic atrophy type 12 (OPA12) manifests as decreased visual acuity (variable but frequently ranging from 0.2/10 to 2/10), photophobia, and impaired color vision. Ophthalmologic findings are optic nerve pallor and highly reduced retinal nerve fiber layer on optical coherence tomography. Although affected individuals do not present with ataxia, some may exhibit sensorineural hearing loss, neurodevelopmental disorders, dystonia, and spasticity. The diagnoses of SCA28 and OPA12 are established in a proband with suggestive findings and a heterozygous pathogenic variant in AFG3L2 identified by molecular genetic testing. The diagnoses of SPAX5 and AFG3L2-SCAR are established in a proband with suggestive findings and biallelic pathogenic variants in AFG3L2 identified by molecular genetic testing. Treatment of manifestations: Supportive care to improve quality of life, maximize function, and reduce complications is recommended. This ideally involves multidisciplinary care by specialists in relevant fields including neurologists (to address pharmacologic treatment of myoclonic epilepsy, spasticity, movement disorders); occupational therapists (to optimize activities of daily living and home safety); physiatrists and physical therapists (to help maintain independence and mobility); nutritionists and feeding therapy programs (to assess the risks of aspiration and need for gastrostomy tube placement for those with dysphagia); speech-language therapists (to address communication for individuals who have expressive language difficulties), ophthalmologists (to consider surgery for ptosis); low vision clinics (for those with optic atrophy); and social workers and psychologists (depending on any cognitive or psychologic manifestations). Surveillance: Routinely scheduled follow-up appointments with treating clinicians are recommended to monitor existing manifestations, the individual's response to supportive care, and the emergence of new manifestations. Agents/circumstances to avoid: Alcohol consumption and sedatives such as benzodiazepines that may worsen gait ataxia and coordination. Carbamazepine and phenytoin may exacerbate myoclonus in SPAX5. SCA28 and OPA12 are inherited in an autosomal dominant manner. AFG3L2-SCAR and SPAX5 are inherited in an autosomal recessive manner. Autosomal dominant inheritance: Most individuals diagnosed with SCA28, and some individuals diagnosed with OPA12, have an affected parent. Some individuals diagnosed with an autosomal dominant AFG3L2-related neurologic disorder have the disorder as the result of a de novo pathogenic variant. Each child of an individual with an autosomal dominant AFG3L2-related neurologic disorder has a 50% risk of inheriting the pathogenic variant. If the reproductive partner of an individual with an autosomal dominant AFG3L2-related neurologic disorder also has an AFG3L2 pathogenic variant, offspring are at risk of inheriting biallelic pathogenic variants and having an autosomal recessive AFG3L2-related neurologic disorder. Once the AFG3L2 pathogenic variant has been identified in an affected family member, predictive testing for at-risk relatives and prenatal/preimplantation genetic testing are possible. Autosomal recessive inheritance: The parents of a child with an autosomal recessive AFG3L2-related neurologic disorder are presumed to be heterozygous for an AFG3L2 pathogenic variant. If both parents are known to be heterozygous for an AFG3L2 pathogenic variant, each sib of an affected individual has at conception a 25% chance of inheriting biallelic pathogenic variants and being affected, a 50% chance of being heterozygous, and a 25% chance of inheriting neither of the familial AFG3L2 pathogenic variants. Heterozygous family members of an individual with an autosomal recessive AFG3L2-related neurologic disorder are typically asymptomatic and the risk of developing an AFG3L2-related neurologic disorder appears to be low. Once the AFG3L2 pathogenic variants has been identified in an affected family member, carrier testing for at-risk relatives and prenatal/preimplantation genetic testing are possible.

AFG3L2 is a mitochondrial protease exerting protein quality control in the inner mitochondrial membrane. Heterozygous AFG3L2 mutations cause spinocerebellar ataxia type 28 (SCA28) or dominant optic atrophy type 12 (DOA12), while biallelic AFG3L2 mutations result in the rare and severe spastic ataxia type 5 (SPAX5). The clinical spectrum of SPAX5 includes childhood-onset cerebellar ataxia, spasticity, dystonia and myoclonic epilepsy. We previously reported that the absence or mutation of AFG3L2 leads to the accumulation of mitochondria-encoded proteins, causing the overactivation of the stress-sensitive protease OMA1, which over-processes OPA1, leading to mitochondrial fragmentation. Recently, OMA1 has been identified as the pivotal player communicating mitochondrial stress to the cytosol via a pathway involving the inner mitochondrial membrane protein DELE1 and the cytosolic kinase HRI, thus eliciting the integrated stress response. In general, the integrated stress response reduces global protein synthesis and drives the expression of cytoprotective genes that allow cells to endure proteotoxic stress. However, the relevance of the OMA1-DELE1-HRI axis in vivo, and especially in a human CNS disease context, has been poorly documented thus far. In this work, we demonstrated that mitochondrial proteotoxicity in the absence/mutation of AFG3L2 activates the OMA1-DELE1-HRI pathway eliciting the integrated stress response. We found enhanced OMA1-dependent processing of DELE1 upon depletion of AFG3L2. Also, in both skin fibroblasts from SPAX5 patients (including a novel case) and in the cerebellum of Afg3l2-/- mice we detected increased phosphorylation of the α-subunit of the eukaryotic translation initiation factor 2 (eIF2α), increased levels of ATF4 and strong upregulation of its downstream targets (Chop, Chac1, Ppp1r15a and Ffg21). Silencing of DELE1 or HRI in SPAX5 fibroblasts (where OMA1 is overactivated at basal state) reduces eIF2α phosphorylation and affects cell growth. In agreement, pharmacological potentiation of integrated stress response via Sephin-1, a drug that selectively inhibits the stress-induced eIF2alpha phosphatase GADD34 (encoded by Ppp1r15a), improved cell growth of SPAX5 fibroblasts and cell survival and dendritic arborization ex vivo in primary Afg3l2-/- Purkinje neurons. Notably, Sephin-1 treatment in vivo extended the lifespan of Afg3l2-/- mice, improved Purkinje neuron morphology, mitochondrial ultrastructure and respiratory capacity. These data indicate that activation of the OMA1-DELE1-HRI pathway is protective in the context of SPAX5. Pharmacological tuning of the integrated stress response may represent a future therapeutic strategy for SPAX5 and other cerebellar ataxias caused by impaired mitochondrial proteostasis.

AFG3L2 is a zinc metalloprotease and an ATPase localized in an inner mitochondrial membrane involved in mitochondrial quality control of several nuclear- and mitochondrial-encoded proteins. Mutations in AFG3L2 lead to diseases like slow progressive ataxia, which is a neurological disorder. This review delineates the cellular functions of AFG3L2 and its dysfunction that leads to major clinical outcomes, which include spinocerebellar ataxia type 28, spastic ataxia type 5, and optic atrophy type 12. It summarizes all relevant AFG3L2 mutations associated with the clinical outcomes to understand the detailed mechanisms attributable to its structure-related multifaceted roles in proteostasis and quality control. We face early diagnostic challenges of ataxia and optic neuropathy due to asymptomatic parents and variable clinical manifestations due to heterozygosity/homozygosity of AFG3L2 mutations. This review intends to promote AFG3L2 as a putative prognostic or diagnostic marker.

The AFG3L2 gene encodes AFG3-like protein 2, which is a subunit of human mitochondrial ATPases associated with various cellular protease activities (m-AAA). The clinical spectrum of AFG3L2 mutations is broad. Dominant AFG3L2 mutations can cause autosomal dominant spinocerebellar ataxia type 28 (SCA28), whereas biallelic AFG3L2 mutations may lead to spastic ataxia 5 (SPAX5). However, the role of AFG3L2 mutations in autosomal recessive spinocerebellar ataxia (SCAR) remains elusive. The aim of this study is to delineate the clinical features and spectrum of AFG3L2 mutations in a Taiwanese cohort with cerebellar ataxia. Mutational analyses of AFG3L2 were carried out by targeted resequencing in a cohort of 133 unrelated patients with molecularly undetermined cerebellar ataxia. We identified one single patient carrying compound heterozygous mutations in AFG3L2, p.[R632*];[V723M] (c.[1894C > T];[2167G > A]). The patient has suffered from apparently sporadic and slowly progressive cerebellar ataxia, ptosis, and ophthalmoparesis since age 55 years. These findings expand the clinical spectrum of AFG3L2 mutations and suggest a new subtype of late-onset SCAR caused by biallelic AFG3L2 mutations.