CLPP deficiency ameliorates neurodegeneration caused by impaired mitochondrial protein synthesis.

Mitochondria are essential organelles found in every eukaryotic cell, required to convert food into usable energy. Therefore, it is not surprising that mutations in either mtDNA or nuclear DNA-encoded genes of mitochondrial proteins cause diseases affecting the oxidative phosphorylation system, which are heterogeneous from a clinical, genetic, biochemical and molecular perspective and can affect patients at any age. Despite all this, it is surprising that our understanding of the mechanisms governing mitochondrial gene expression and its associated pathologies remain superficial and therapeutic interventions largely unexplored. We recently showed that loss of the mitochondrial matrix protease caseinolytic protease proteolytic subunit (CLPP) ameliorates phenotypes in cells characterized by defects in oxidative phosphorylation maintenance. Here, we build upon this finding by showing that CLPP depletion is indeed beneficial in vivo for various types of neuronal populations, including Purkinje cells in the cerebellum and cortical and hippocampal neurons in the forebrain, as it strongly improves distinct phenotypes of mitochondria encephalopathy, driven by the deficiency of the mitochondrial aspartyl tRNA synthase DARS2. In the absence of CLPP, neurodegeneration of DARS2-deficient neurons is delayed as they present milder oxidative phosphorylation dysfunction. This in turn leads to a decreased neuroinflammatory response and significantly improved motor functions in both double-deficient models (Purkinje cell-specific or forebrain neuron-specific Dars2/Clpp double knockout mice). We propose that diminished turnover of respiratory complex I caused by the loss of CLPP is behind the improved phenotype in Dars2/Clpp double knockout animals, even though this intervention might not restore respiratory complex I activity but rather improve mitochondrial cristae morphology or help maintain the NAD+/NADH ratio inside mitochondria. These results also open the possibility of targeting CLPP activity in many other mitochondrial encephalopathies characterized by respiratory complex I instability.

Intra-familial phenotypic heterogeneity in a Sudanese family with DARS2-related leukoencephalopathy, brainstem and spinal cord involvement and lactate elevation: a case report.

BACKGROUND: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL, OMIM #611105) is a genetic disease of the central nervous system characterized by lower limb spasticity, cerebellar ataxia and involvement of the dorsal column. The disease is caused by mutations in the DARS2 gene but has never been reported in sub-Saharan Africa so far. CASE PRESENTATION: Two siblings, aged 18 years and 15 years, from a consanguineous family presented with pyramidal signs and symptoms since infancy and developmental delay. Whole exome sequencing of the proband identified two compound heterozygous variants (NM_018122.4:c.1762C > G and c.563G > A) in DARS2. Sanger sequencing confirmed the presence of the mutations and their segregation in trans in both patients and in their elder sister (aged 20 years), who showed only brisk reflexes and mild lower limb spasticity. Surprisingly, in contrast to her subtle clinical presentation, the elder sister had abnormal MRI features and serum lactate levels comparable to her ill sisters. CONCLUSION: This report illustrates intra-familial phenotypic variation in LBSL and provides an example of a marked dissociation between the clinical and radiological phenotypes of the disease. This may have implications for the detection of mutation carriers in LBSL.

Neonatal mitochondrial leukoencephalopathy with brain and spinal involvement and high lactate: expanding the phenotype of ISCA2 gene mutations.

A homoallelic missense founder mutation of the iron-sulfur cluster assembly 2 (ISCA2) gene has been recently reported in six cases affected by an autosomal recessive infantile neurodegenerative mitochondrial disorder. We documented a case of a 2-month-old girl presenting with severe hypotonia and nystagmus, who rapidly deteriorated and died at the age of three months. Increased cerebral spinal fluid level of lactate, documented also at the brain spectroscopy, involvement of the cortex, restricted diffusion of white and gray matter abnormalities, sparing of the corpus callosum and extensive involvement of the spinal cord were observed. Her clinical presenting features and course as well as some neuroradiological findings mimicked those of early-onset leukoencephalopathy with brainstem and spinal cord involvement and high brain lactate (LBSL). The analysis of the mitochondrial respiratory chain function showed a reduced activity of complexes II and IV. The girl harboured two heterozygous mutations in the ISCA2 gene. A comprehensive review of the literature and a comparison with the cases of early onset LBSL enabled us to highlight significant differences in the clinical, biochemical and neuroradiological phenotype between the two conditions, which also emerged from the comparison with the other 6 reported cases of ISCA2 gene mutation previously reported. In summary, this represents the second report ever published associating ISCA2 gene mutation with a mitochondrial leukoencephalopathy, with a different genetic mechanism to the previous cases. Molecular analysis of ISCA2 should be included in the genetic panel for the diagnosis of early onset mitochondrial leukoencephalopathies.

Mutation of the human mitochondrial phenylalanine-tRNA synthetase causes infantile-onset epilepsy and cytochrome c oxidase deficiency.

Mitochondrial aminoacyl-tRNA synthetases (aaRSs) are essential enzymes in protein synthesis since they charge tRNAs with their cognate amino acids. Mutations in the genes encoding mitochondrial aaRSs have been associated with a wide spectrum of human mitochondrial diseases. Here we report the identification of pathogenic mutations (a partial genomic deletion and a highly conserved p. Asp325Tyr missense variant) in FARS2, the gene encoding mitochondrial phenylalanyl-tRNA synthetase, in a patient with early-onset epilepsy and isolated complex IV deficiency in muscle. The biochemical defect was expressed in myoblasts but not in fibroblasts and associated with decreased steady state levels of COXI and COXII protein and reduced steady state levels of the mt-tRNA(Phe) transcript. Functional analysis of the recombinant mutant p. Asp325Tyr FARS2 protein showed an inability to bind ATP and consequently undetectable aminoacylation activity using either bacterial tRNA or human mt-tRNA(Phe) as substrates. Lentiviral transduction of cells with wildtype FARS2 restored complex IV protein levels, confirming that the p.Asp325Tyr mutation is pathogenic, causing respiratory chain deficiency and neurological deficits on account of defective aminoacylation of mt-tRNA(Phe).

Early-onset leukoencephalopathy due to a homozygous missense mutation in the DARS2 gene.

Mutations in the DARS2 gene are known to cause leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL), a rare autosomal recessive neurological disorder. It was originally described as juvenile-onset slowly progressive ataxia and spasticity, but recent reports suggest a broader clinical spectrum. Most patients were found to carry compound heterozygous DARS2 mutations, and only very few patients with homozygous mutations have been described so far. We present here an 8-month-old boy carrying a homozygous missense mutation in DARS2 who clinically showed severe neurological deterioration after a respiratory tract infection, followed by an almost complete remission of symptoms. This report further extends the knowledge about the clinical and molecular genetic spectrum of LBSL.

Identification of a Novel Indel Variant in the DARS2 Gene in Russian Patients with Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation.

BACKGROUND: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation is an inherited disease caused by pathogenic biallelic variants in the gene DARS2, which encodes mitochondrial aspartyl-tRNA synthetase. This disease is characterized by slowly progressive spastic gait, cerebellar symptoms, and leukoencephalopathy with brainstem and spinal cord involvement. CASE PRESENTATION: Peripheral blood samples were collected from four patients from four unrelated families to extract genomic DNA. All patients underwent partial exon analysis of the DARS2 gene using Sanger sequencing, which detected the c.228-21_228-20delinsC variant in a heterozygous state. Further DNA from three patients was analyzed using a next-generation sequencing-based custom AmpliSeq™ panel for 59 genes associated with leukodystrophies, and one of the patients underwent whole genome sequencing. We identified a novel pathogenic variant c.1675-1256_*115delinsGCAACATTTCGGCAACATTCCAACC in the DARS2 gene. Three patients (patients 1, 2, and 4) had slowly progressive cerebellar ataxia, and two patients (patients 1 and 2) had spasticity. In addition, two patients (patients 2 and 4) showed signs of axonal neuropathy, such as decreased tendon reflexes and loss of distal sensitivity. Three patients (patients 1, 2, and 3) also had learning difficulties. It should be noted the persistent presence of characteristic changes in brain MRI in all patients, which emphasizes its importance as the main diagnostic tool for suspicion and subsequent confirmation of LBSL. Conclusions: We found a novel indel variant in the DARS2 gene in four patients with LBSL and described their clinical and genetic characteristics. These results expand the mutational spectrum of LBSL and aim to improve the laboratory diagnosis of this form of leukodystrophy.

Post-Vaccination/Post-COVID Immune-Mediated Demyelination of the Brain and Spinal Cord: A Novel Neuroimaging Finding.

Introduction: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes coronavirus disease 2019 (COVID-19), which not only produces respiratory symptoms but is known to involve almost every system, and its neuroinvasive properties have been well demonstrated throughout the pandemic. Also, to combat the pandemic, there was rapid development and induction of various vaccination drives, following which many adverse events following immunization (AEFIs) have been reported, which include neurological complications as well. Method: We present a series of three cases, post vaccination, with and without a history of COVID illness that showed remarkably similar findings on magnetic resonance imaging (MRI). Result: A 38-year-old male presented with complaints of weakness of the bilateral lower limbs with sensory loss and bladder disturbance a day after receiving his first dose of ChadOx1 nCoV-19 (COVISHIELD) vaccine. A 50-year-old male with hypothyroidism characterized by autoimmune thyroiditis and impaired glucose tolerance experienced difficulty in walking 11.5 weeks after being administered with COVID vaccine (COVAXIN). A 38-year-old male presented with subacute onset progressive symmetric quadriparesis 2 months after their first dose of a COVID vaccine. The patient also had sensory ataxia, and his vibration sensation was impaired below C7. All three patients had typical pattern of involvement of the brain and spine on MRI with signal changes in bilateral corticospinal tracts, trigeminal tracts in the brain, and both lateral and posterior columns in the spine. Conclusion: This pattern of brain and spine involvement on MRI is a novel finding and is likely a result of post-vaccination/post-COVID immune-mediated demyelination.

Perinatal Manifestations of DARS2-Associated Leukoencephalopathy With Brainstem and Spinal Cord Involvement and Lactate Elevation (LBSL).

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a progressive disorder associated with deficiency of mitochondrial aspartyl-tRNA synthetase, a homodimer encoded by the gene DARS2. There is a wide range in age of onset of symptoms, typically from childhood to adulthood, with very few cases of infantile onset disease reported. We report a child at age 10 years with perinatal onset of symptoms evidenced by congenital microcephaly with progression to severe but non-lethal epileptic encephalopathy and spastic quadriplegia. A comprehensive epilepsy focused gene panel performed as a trio with parents detected a novel homozygous DARS2 variant. This variant is located at the dimer interface in a critical catalytic domain and is expected to result in markedly reduced enzyme activity which likely explains the severe and early onset symptoms in this case.

Leukoencephalopathy with Brainstem and Spinal Cord Involvement and Lactate Elevation: A Novel DARS2 Mutation and Intra-Familial Heterogeneity.

BACKGROUND: Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is characterized by slowly progressive spastic gait, cerebellar symptoms, and posterior cord dysfunction. DARS2, which encodes mitochondrial aspartyl tRNA synthase, is associated with the rare disease. CASES: The proband had gait disturbance since age 56, while her younger brother had the gait problem since his 20s and needed cane-assistance at age 45. Both cases showed typical demyelinating features of LBSL on the magnetic resonance imaging (MRI) involving the periventricular white matter, brainstem, cerebellum and spinal cord. Sequencing of both cases showed compound heterozygous mutations: c.228-16C>A and c.508C>T in DARS2. The c.228-16C>A is a common mutation in splicing site of intron 2, which causes alternative splicing defect of exon 3, while the c.508C>T at the exon 6 is novel. Our patients are unique in the relative late onset and the apparent difference in disease progression. LITERATURE REVIEW: Literatures from PubMed were reviewed. Five families showed intra-familial heterogeneity on age at onset or clinical severity. CONCLUSION: We identified a family of LBSL with compound heterozygous mutations, and c.508C>T at the exon 6 is a novel one. Clinical heterogeneity was observed in the family and other literatures. Further research for underlying mechanism is required.

A New DARS2 Mutation Discovered in an Adult Patient.

We report a case of an adult patient suffering from leukoencephalopathy with brainstem and spinal cord involvement and elevated white matter lactate (LBSL) caused by a DARS2 polymorphism. DARS2 mutation was identified by combining MRI and genetic analysis. Our patient was affected by compound heterozygosity for a pathogenic mutation and a common variant, but with reduced aspartyl-tRNA synthetase activity. Brain and spinal cord magnetic resonance imaging revealed extensive white matter abnormalities; spectroscopy revealed no lactate elevation. A new compound heterozygous DARS2 variant combined with a polymorphism in the other allele in an adult patient with LBSL was identified, resulting in reduced DARS2 activity. This combination is rare and has consequences on how we should consider benign variant polymorphisms in the future.

Neuronal ablation of mt-AspRS in mice induces immune pathway activation prior to severe and progressive cortical and behavioral disruption.

Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is a rare, slowly progressive white matter disease caused by mutations in the mitochondrial aspartyl-tRNA synthetase (mt-AspRS, or DARS2). While patients show characteristic MRI T2 signal abnormalities throughout the cerebral white matter, brainstem, and spinal cord, the phenotypic spectrum is broad and a multitude of gene variants have been associated with the disease. Here, Dars2 disruption in CamKIIα-expressing cortical and hippocampal neurons results in slowly progressive increases in behavioral activity at five months, and culminating by nine months as severe brain atrophy, behavioral dysfunction, reduced corpus callosum thickness, and microglial morphology indicative of neuroinflammation. Interestingly, RNAseq based gene expression studies performed prior to the presentation of this severe phenotype reveal the upregulation of several pathways involved in immune activation, cytokine production and signaling, and defense response regulation. RNA transcript analysis demonstrates that activation of immune and cell stress pathways are initiated in advance of a behavioral phenotype and cerebral deficits. An understanding of these pathways and their contribution to significant neuronal loss in CamKII-Dars2 deficient mice may aid in deciphering mechanisms of LBSL pathology.

Mitochondrial aminoacyl-tRNA synthetase disorders: an emerging group of developmental disorders of myelination.

BACKGROUND: The mitochondrial aminoacyl-tRNA synthetase proteins (mt-aaRSs) are a group of nuclear-encoded enzymes that facilitate conjugation of each of the 20 amino acids to its cognate tRNA molecule. Mitochondrial diseases are a large, clinically heterogeneous group of disorders with diverse etiologies, ages of onset, and involved organ systems. Diseases related to mt-aaRS mutations are associated with specific syndromes that affect the central nervous system and produce highly characteristic MRI patterns, prototypically the DARS2, EARS, and AARS2 leukodystrophies, which are caused by mutations in mitochondrial aspartyl-tRNA synthetase, mitochondria glutamate tRNA synthetase, and mitochondrial alanyl-tRNA synthetase, respectively. BODY: The disease patterns emerging for these leukodystrophies are distinct in terms of the age of onset, nature of disease progression, and predominance of involved white matter tracts. In DARS2 and EARS2 disorders, earlier disease onset is typically correlated with more significant brain abnormalities, rapid neurological decline, and greater disability. In AARS2 leukodystrophy cases reported thus far, there is nearly invariable progression to severe disability and atrophy of involved brain regions, often within a decade. Although most mutations are compound heterozygous inherited in an autosomal recessive fashion, homozygous variants are found in each disorder and demonstrate high phenotypic variability. Affected siblings manifest disease on a wide spectrum. CONCLUSION: The syndromic nature and selective vulnerability of white matter tracts in these disorders suggests there may be a shared mechanism of mitochondrial dysfunction to target for study. There is evidence that the clinical variability and white matter tract specificity of each mt-aaRS leukodystrophy depend on both canonical and non-canonical effects of the mutations on the process of mitochondrial translation. Furthermore, different sensitivities to the mt-aaRS mutations have been observed based on cell type. Most mutations result in at least partial retention of mt-aaRS enzyme function with varied effects on the mitochondrial respiratory chain complexes. In EARS2 and AARS2 cells, this appears to result in cumulative impairment of respiration. Mt-aaRS mutations may also affect alternative biochemical pathways such as the integrated stress response, a homeostatic program in eukaryotic cells that typically confers cytoprotection, but can lead to cell death when abnormally activated in response to pathologic states. Systematic review of this group of disorders and further exploration of disease mechanisms in disease models and neural cells are warranted.

The first pediatric case of leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) from Turkey.

Çavusoglu D, Olgaç-Dündar N, Öztekin Ö, Özdemir TR, Arican P, Gençpinar P. The first pediatric case of leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) from Turkey. Turk J Pediatr 2018; 60: 216-220. Leukoencephalopathy with brainstem and spinal cord involvement and lactate elevation (LBSL) is defined as an autosomal recessive inheritance disorder characterized by slowly progressive cerebellar, pyramidal and dorsal column dysfunction. The diagnosis is based on specific magnetic resonance imaging abnormalities (MRI) in the cerebral and cerebellar white matter and selective involvement of white matter tracts in the brain stem and spinal cord. LBSL is caused by mutations in the DARS2 gene which encodes the mitochondrial aspartyl-tRNA synthetase. Herein, we report the first pediatric case from Turkey with a typical MRI course of LBSL associated with a compound heterozygous mutation in DARS2 gene.