Bi-allelic mutations of LONP1 encoding the mitochondrial LonP1 protease cause pyruvate dehydrogenase deficiency and profound neurodegeneration with progressive cerebellar atrophy.

LonP1 is crucial for maintaining mitochondrial proteostasis and mitigating cell stress. We identified a novel homozygous missense LONP1 variant, c.2282 C > T, (p.Pro761Leu), by whole-exome and Sanger sequencing in two siblings born to healthy consanguineous parents. Both siblings presented with stepwise regression during infancy, profound hypotonia and muscle weakness, severe intellectual disability and progressive cerebellar atrophy on brain imaging. Muscle biopsy revealed the absence of ragged-red fibers, however, scattered cytochrome c oxidase-negative staining and electron dense mitochondrial inclusions were observed. Primary cultured fibroblasts from the siblings showed normal levels of mtDNA and mitochondrial transcripts, and normal activities of oxidative phosphorylation complexes I through V. Interestingly, fibroblasts of both siblings showed glucose-repressed oxygen consumption compared to their mother, whereas galactose and palmitic acid utilization were similar. Notably, the siblings' fibroblasts had reduced pyruvate dehydrogenase (PDH) activity and elevated intracellular lactate:pyruvate ratios, whereas plasma ratios were normal. We demonstrated that in the siblings' fibroblasts, PDH dysfunction was caused by increased levels of the phosphorylated E1α subunit of PDH, which inhibits enzyme activity. Blocking E1α phosphorylation activated PDH and reduced intracellular lactate concentrations. In addition, overexpressing wild-type LonP1 in the siblings' fibroblasts down-regulated phosphoE1α. Furthermore, in vitro studies demonstrated that purified LonP1-P761L failed to degrade phosphorylated E1α, in contrast to wild-type LonP1. We propose a novel mechanism whereby homozygous expression of the LonP1-P761L variant leads to PDH deficiency and energy metabolism dysfunction, which promotes severe neurologic impairment and neurodegeneration.

Mitochondrial PITRM1 peptidase loss-of-function in childhood cerebellar atrophy.

OBJECTIVE: To identify the genetic basis of a childhood-onset syndrome of variable severity characterised by progressive spinocerebellar ataxia, mental retardation, psychotic episodes and cerebellar atrophy. METHODS: Identification of the underlying mutations by whole exome and whole genome sequencing. Consequences were examined in patients' cells and in yeast. RESULTS: Two brothers from a consanguineous Palestinian family presented with progressive spinocerebellar ataxia, mental retardation and psychotic episodes. Serial brain imaging showed severe progressive cerebellar atrophy. Whole exome sequencing revealed a novel mutation: pitrilysin metallopeptidase 1 (PITRM1) c.2795C>T, p.T931M, homozygous in the affected children and resulting in 95% reduction in PITRM1 protein. Whole genome sequencing revealed a chromosome X structural rearrangement that also segregated with the disease. Independently, two siblings from a second Palestinian family presented with similar, somewhat milder symptoms and the same PITRM1 mutation on a shared haplotype. PITRM1T931M carrier frequency was 0.027 (3/110) in the village of the first family evaluated, and 0/300 among Palestinians from other locales. PITRM1 is a mitochondrial matrix enzyme that degrades 10-65 amino acid oligopeptides, including the mitochondrial fraction of amyloid-beta peptide. Analysis of peptide cleavage activity by the PITRM1T931M protein revealed a significant decrease in the degradation capacity specifically of peptides ≥40 amino acids. CONCLUSION: PITRM1T931M results in childhood-onset recessive cerebellar pathology. Severity of PITRM1-related disease may be affected by the degree of impairment in cleavage of mitochondrial long peptides. Disruption and deletion of X linked regulatory segments may also contribute to severity.

Human aminoacyl-tRNA synthetases in diseases of the nervous system.

Aminoacyl-tRNA synthetases (AaRSs) are ubiquitously expressed enzymes that ensure accurate translation of the genetic information into functional proteins. These enzymes also execute a variety of non-canonical functions that are significant for regulation of diverse cellular processes and that reside outside the realm of protein synthesis. Associations between faults in AaRS-mediated processes and human diseases have been long recognized. Most recent research findings strongly argue that 10 cytosolic and 14 mitochondrial AaRSs are implicated in some form of pathology of the human nervous system. The advent of modern whole-exome sequencing makes it all but certain that similar associations between the remaining 15 ARS genes and neurologic illnesses will be defined in future. It is not surprising that an intense scientific debate about the role of translational machinery, in general, and AaRSs, in particular, in the development and maintenance of the healthy human neural cell types and the brain is sparked. Herein, we summarize the current knowledge about causative links between mutations in human AaRSs and diseases of the nervous system and briefly discuss future directions.

[The role of ATM kinase in neurodegeneration]. / Znaczenie kinazy ATM w procesach neurodegeneracyjnych.

Neurodegenerative diseases represent a major challenge for modern medicine. Despite many years of research, no effective neuroprotective therapy has been proposed. Ataxia telangiectasia (A-T) is rare disease, which is caused by a mutation of the ATM protein. Cerebellar degeneration is the main symptom of the A-T. The kinase ATM, inter alia is involved in the repair of DNA damage, cell cycle regulation and the control of apoptosis. In recent years the presence of that kinase in the cytoplasm has been demonstrated. This led to the discovery of its participation in the regulation of metabolic processes, homeostasis mitochondrial oxidative stress response or modulation of synaptic function. The pleiotropic effect of ATM kinase requires effective control exercised by, inter alia, proteins having specific binding motifs this kinase, such as ATMIN and NBS1. The regulation of prosurvival processes which are controlled by ATM kinase, may prove an attractive therapeutic strategy in treatment of neurodegenerative diseases.

The ciliopathy-associated protein homologs RPGRIP1 and RPGRIP1L are linked to cilium integrity through interaction with Nek4 serine/threonine kinase.

Recent studies have established ciliary dysfunction as the underlying cause of a broad range of multi-organ phenotypes, known as 'ciliopathies'. Ciliopathy-associated proteins have a common site of action in the cilium, however, their overall importance for ciliary function differs, as implied by the extreme variability in ciliopathy phenotypes. The aim of this study was to gain more insight in the function of two ciliopathy-associated protein homologs, RPGR interacting protein 1 (RPGRIP1) and RPGRIP1-like protein (RPGRIP1L). Mutations in RPGRIP1 lead to the eye-restricted disease Leber congenital amaurosis, while mutations in RPGRIP1L are causative for Joubert and Meckel syndrome, which affect multiple organs and are at the severe end of the ciliopathy spectrum. Using tandem affinity purification in combination with mass spectrometry, we identified Nek4 serine/threonine kinase as a prominent component of both the RPGRIP1- as well as the RPGRIP1L-associated protein complex. In ciliated cells, this kinase localized to basal bodies, while in ciliated organs, the kinase was predominantly detected at the ciliary rootlet. Down-regulation of NEK4 in ciliated cells led to a significant decrease in cilium assembly, pointing to a role for Nek4 in cilium dynamics. We now hypothesize that RPGRIP1 and RPGRIP1L function as cilium-specific scaffolds that recruit a Nek4 signaling network which regulates cilium stability. Our data are in line with previously established roles in the cilium of other members of the Nek protein family and define NEK4 as a ciliopathy candidate gene.

Cerebellar abnormalities in purine nucleoside phosphorylase deficient mice.

Inherited defects in purine nucleoside phosphorylase (PNP) cause severe T cell immunodeficiency and progressive neurological dysfunction, yet little is known about the effects of PNP deficiency on the brain. PNP-KO mice display metabolic and immune anomalies similar to those observed in patients. Our objectives were to characterize brain abnormalities in PNP-KO mice and determine whether restoring PNP activity prevents these abnormalities. We analyzed structural brain defects in PNP-KO mice by magnetic resonance imaging, while assessing motor deficits using the accelerating rotarod and stationary balance beam tests. We detected morphological abnormalities and apoptosis in the cerebellum of PNP-KO mice by hematoxylin and eosin, electron microscopy, TUNEL and activated caspase 3 staining. We treated PNP-KO mice with PNP fused to the HIV-TAT protein transduction domain (TAT-PNP) from birth or from 4 weeks of age. Magnetic resonance imaging revealed a smaller than normal cerebellum in PNP-KO mice. PNP-KO mice displayed motor abnormalities including rapid fall from the rotating rod and frequent slips from the balance beam. The cerebellum of PNP-KO mice contained reduced purkinje cells (PC), which were irregular in shape and had degenerated dendrites. PC from the cerebellum of PNP-KO mice, expanded ex vivo, demonstrated increased apoptosis, which could be corrected by supplementing cultures with TAT-PNP. TAT-PNP injections restored PNP activity in the cerebellum of PNP-KO mice. TAT-PNP from birth, but not treatment initiated at 4 weeks of age, prevented the cerebellar PC damage and motor deficits. We conclude that PNP deficiency cause cerebellar abnormalities, including PC damage and progressive motor deficits. TAT-PNP treatment from birth can prevent the neurological abnormalities in PNP-KO mice.

Genetic interaction between the m-AAA protease isoenzymes reveals novel roles in cerebellar degeneration.

The mitochondrial m-AAA protease has a crucial role in axonal development and maintenance. Human mitochondria possess two m-AAA protease isoenzymes: a hetero-oligomeric complex, composed of paraplegin and AFG3L2 (Afg3 like 2), and a homo-oligomeric AFG3L2 complex. Loss of function of paraplegin (encoded by the SPG7 gene) causes hereditary spastic paraplegia, a disease characterized by retrograde degeneration of cortical motor axons. Spg7(-/-) mice show a late-onset degeneration of long spinal and peripheral axons with accumulation of abnormal mitochondria. In contrast, Afg3l2(Emv66/Emv66) mutant mice, lacking the AFG3L2 protein, are affected by a severe neuromuscular phenotype, due to defects in motor axon development. The role of the homo-oligomeric m-AAA protease and the extent of cooperation and redundancy between the two isoenzymes in adult neurons are still unclear. Here we report an early-onset severe neurological phenotype in Spg7(-/-) Afg3l2(Emv66/+) mice, characterized by loss of balance, tremor and ataxia. Spg7(-/-) Afg3l2(Emv66/+) mice display acceleration and worsening of the axonopathy observed in paraplegin-deficient mice. In addition, they show prominent cerebellar degeneration with loss of Purkinje cells and parallel fibers, and reactive astrogliosis. Mitochondria from affected tissues are prone to lose mt-DNA and have unstable respiratory complexes. At late stages, neurons contain structural abnormal mitochondria defective in COX-SDH reaction. Our data demonstrate genetic interaction between the m-AAA isoenzymes and suggest that different neuronal populations have variable thresholds of susceptibility to reduced levels of the m-AAA protease. Moreover, they implicate impaired mitochondrial proteolysis as a novel pathway in cerebellar degeneration.

Genetic background conversion ameliorates semi-lethality and permits behavioral analyses in cystathionine beta-synthase-deficient mice, an animal model for hyperhomocysteinemia.

Cystathionine beta-synthase-deficient mice (Cbs(-/-)) exhibit several pathophysiological features similar to hyperhomocysteinemic patients, including endothelial dysfunction and hepatic steatosis. Heterozygous mutants (Cbs(+/-)) on the C57BL/6J background are extensively analyzed in laboratories worldwide; however, detailed analyses of Cbs(-/-) have been hampered by the fact that they rarely survive past the weaning age probably due to severe hepatic dysfunction. We backcrossed the mutants with four inbred strains (C57BL/6J(Jcl), BALB/cA, C3H/HeJ and DBA/2J) for seven generations, and compared Cbs(-/-) phenotypes among the different genetic backgrounds. Although Cbs(-/-) on all backgrounds were hyperhomocysteinemic/hypermethioninemic and suffered from lipidosis/hepatic steatosis at 2 weeks of age, >30% of C3H/HeJ-Cbs(-/-) survived over 8 weeks whereas none of DBA/2J-Cbs(-/-) survived beyond 5 weeks. At 2 weeks, serum levels of total homocysteine and triglyceride were lowest in C3H/HeJ-Cbs(-/-). Adult C3H/HeJ-Cbs(-/-) survivors showed hyperhomocysteinemia but escaped hypermethioninemia, lipidosis and hepatic steatosis. They appeared normal in general behavioral tests but showed cerebellar malformation and impaired learning ability in the passive avoidance step-through test, and required sufficient dietary supplementation of cyst(e)ine for survival, demonstrating the essential roles of cystathionine beta-synthase in the central nervous system function and cysteine biosynthesis. Our C3H/HeJ-Cbs(-/-) mice could be useful tools for investigating clinical symptoms such as mental retardation and thromboembolism that are found in homocysteinemic patients.

PTPRR, cerebellum, and motor coordination.

Tyrosine phosphorylation is a powerful mechanism of modulation for proliferation, differentiation, and functioning of neurons. The protein products of the neuronal mouse gene PTPRR are physiological regulators of mitogen-activated protein kinase (MAPK) activities. PTPRR(-/-) mice display deficits of motor coordination and balance skills. PTPRR gene orthologues are found in many vertebrates. Recent observations suggest that the human episodic ataxia 2 (EA2) and spinocerebellar ataxia types 6 (SCA6), 12 (SCA12), and 14 (SCA14) might be associated with impaired phosphorylation levels of cerebellum calcium channels and receptors. The concept that MAPK signaling is a key process in tuning synaptic plasticity in cerebellar circuits is now emerging, with numerous implications for understanding cerebellar functions and cerebellar disorders.

Cerebral creatine deficiency syndromes: clinical aspects, treatment and pathophysiology.

Cerebral creatine deficiency syndromes (CCDSs) are a group of inborn errors of creatine metabolism comprising two autosomal recessive disorders that affect the biosynthesis of creatine--i.e. arginine:glycine amidinotransferase deficiency (AGAT; MIM 602360) and guanidinoacetate methyltransferase deficiency (GAMT; MIM 601240)--and an X-linked defect that affects the creatine transporter, SLC6A8 deficiency (SLC6A8; MIM 300036). The biochemical hallmarks of these disorders include cerebral creatine deficiency as detected in vivo by 1H magnetic resonance spectroscopy (MRS) of the brain, and specific disturbances in metabolites of creatine metabolism in body fluids. In urine and plasma, abnormal guanidinoacetic acid (GAA) levels are found in AGAT deficiency (reduced GAA) and in GAMT deficiency (increased GAA). In urine of males with SLC6A8 deficiency, an increased creatine/creatinine ratio is detected. The common clinical presentation in CCDS includes mental retardation, expressive speech and language delay, autistic like behaviour and epilepsy. Treatment of the creatine biosynthesis defects has yielded clinical improvement, while for creatine transporter deficiency, successful treatment strategies still need to be discovered. CCDSs may be responsible for a considerable fraction of children and adults affected with mental retardation of unknown etiology. Thus, screening for this group of disorders should be included in the differential diagnosis of this population. In this review, also the importance of CCDSs for the unravelling of the (patho)physiology of cerebral creatine metabolism is discussed.

A quantitative assessment of the evolution of cerebellar syndrome in children with phosphomannomutase-deficiency (PMM2-CDG).

BACKGROUND: We aim to delineate the progression of cerebellar syndrome in children with phosphomannomutase-deficiency (PMM2-CDG) using the International Cooperative Ataxia Rating Scale (ICARS). We sought correlation between cerebellar volumetry and clinical situation. We prospectively evaluated PMM2-CDG patients aged from 5 to 18 years through ICARS at two different time points set apart by at least 20 months. We reviewed available MRIs and performed volumetric analysis when it was possible. RESULTS: From the eligible 24, four patients were excluded due to severe mental disability (n = 2) and supratentorial lesions (n = 2). Two different ICARS evaluations separated by more than 20 months were available for 14 patients showing an improvement in the cerebellar syndrome: ICARS1: 35.71 versus ICARS2: 30.07 (p < 0.001). When we considered time, we saw an improvement of 2.64 points in the ICARS per year with an SD of 1.97 points (p < 0.001). The ICARS subscales results improved with time, reaching statistical significance in "Posture and gait" (p < 0.001), "Kinetic functions" (p = 0.04) and "Speech abnormalities" (p = 0.045). We found a negative correlation between the ICARS results and total cerebellar volume (r = -0.9, p = 0.037) in a group of five patients with available volumetric study, meaning that the higher the ICARS score, the more severe was the cerebellar atrophy. CONCLUSIONS: Our study shows a stabilization or mild improvement in the cerebellar functions of paediatric PMM2-CDG patients despite cerebellar volume loss. ICARS is a valid scale to quantify the evolution of cerebellar syndrome in PMM2-CDG patients. The availability of ICARS and other reliable and sensitive follow-up tools may prove essential for the evaluation of potential therapies.

Cerebellar degeneration in cattle grazing Solanum bonariense ("Naranjillo") in Western Uruguay.

Cattle in western Uruguay that were eating Solanum bonariense developed periodic episodes of ataxia, hypermetria, hyperesthesia, head and thoracic limb extension, opisthotonus, nystagmus, and falling to the side or backward. Similar clinical signs were experimentally reproduced in cattle by administration of S. bonariense via rumen cannula at a dose of 1,024 g/kg body mass. No significant gross lesions were observed in field cases or experimentally induced cases. Spontaneous and induced histologic lesions were similar and included vacuolation, degeneration, and loss of Purkinje cells. Axonal spheroids, microcavitations, and other changes of wallerian-type degeneration in cerebellar white matter were also observed. Ultrastructural changes included increased number of electron-dense residual storage bodies in membrane-bound vesicles in affected Purkinje cells, and similar vesicles and mitochondria in axonal spheroids. No histologic lesions were detected in the other examined tissues. The Purkinje-cell swelling and vacuolation with subsequent cerebellar degeneration are suggestive of Purkinje-cell specific toxin that produces abnormal lysosome function and cell specific axonal transport. This is the first report of S. bonariense toxicity.

Cerebellar defects in Ca2+/calmodulin kinase IV-deficient mice.

The Ca(2+)/calmodulin-dependent protein kinase CaMKIV was first identified in the cerebellum and has been implicated in nuclear signaling events that control neuronal growth, differentiation, and plasticity. To understand the physiological importance of CaMKIV, we disrupted the mouse Camk4 gene. The CaMKIV null mice displayed locomotor defects consistent with altered cerebellar function. Although the overall cytoarchitecture of the cerebellum appeared normal in the Camk4(-/-) mice, we observed a significant reduction in the number of mature Purkinje neurons and reduced expression of the protein marker calbindin D28k within individual Purkinje neurons. Western immunoblot analyses of cerebellar extracts also established significant deficits in the phosphorylation of cAMP response element-binding protein at serine-133, a proposed target of CaMKIV. Additionally, the absence of CaMKIV markedly altered neurotransmission at excitatory synapses in Purkinje cells. Multiple innervation by climbing fibers and enhanced parallel fiber synaptic currents suggested an immature development of Purkinje cells in the Camk4(-/-) mice. Together, these findings demonstrate that CaMKIV plays key roles in the function and development of the cerebellum.

Spinocerebellar degeneration: hexosaminidase A and B deficiency in two adult sisters.

Two adult sisters had spinocerebellar degeneration. Biochemical studies revealed a very low activity of both fraction A and fraction B of the lysosomal enzyme, hexosaminidase, in serum and leukocytes. A skin biopsy showed lesions suggestive of neuronal storage disease. The disorder seems to be an adult form of GM2 gangliosidosis.

Glutamate dehydrogenase deficiency in patients with olivopontocerebellar atrophy.

Deficiency of glutamate dehydrogenase appears to be associated with a chronic progressive degenerative disorder manifesting parkinsonian extrapyramidal features, ataxia, supranuclear oculomotor dysfunction, a peripheral neuropathy and, in some cases, amyotrophy. The clinical features resemble those of the Dejerine-Thomas type of olivopontocerebellar atrophy. The data suggest autosomal dominant inheritance with low penetrance. Measurement of leukocyte glutamate dehydrogenase should be routinely performed in the evaluation of newly diagnosed or atypical cases of parkinsonism.

Histochemical demonstration of mitochondrial dehydrogenases in developing normal and nervous mutant mouse Purkinje cells.

The mouse mutation nervous (nr), expressed as a recessive, causes a morphologic change in cerebellar Purkinje cell perikaryonal mitochondria during the 2nd postnatal week which is followed by the selective degeneration of most Purkinje cells. In the present study, mitochondrial dehydrogenase activities (succinic acid dehydrogenase, lactic acid dehydrogenase, malic acid dehydrogenase, reduced diphosphopyridine nucleotide diaphorase and succinic semialdehyde dehyrogenase) are found by light microscopy to be similar in the perikarya of normal and nervous Purkinje cells until after mitochondrial rounding has begun to occur, but then staining increases markedly in nervous. Although only a small fraction of mitochondrial functions were examined, those enzymes assayed were present and competent. In a sense, the only mitochondrial abnormalities demonstrated thus far in nervous Purkinje cells are their unusual shape and probable loss of motility.

Hexosaminidase A deficiency in adults.

Deficiency of hexosaminidase A (Hex A) in adults was found in 15 individuals from nine unrelated Ashkenazi families; 14 individuals had neurological symptoms, one was clinically intact. Clinical, biochemical and genetic findings are reported and compared to previously reported cases. The clinical picture varied between and within families and included spinocerebellar, various motor neuron and cerebellar syndromes. Psychosis appeared in 30% of cases. Involvement of three generations was recorded in one family. The phenotype appears too variable to serve as a basis for genetic classification. The level of Hex A activity in serum, leukocytes, and fibroblasts of all 14 patients was in the range of Tay-Sachs disease (TSD) homozygotes when measured by the routine heat-inactivation method. More sensitive and direct methods detected some residual activity. Cultured skin fibroblasts of patients synthesize the alpha and beta chain precursors of Hex A of the same molecular weight as that synthesized by normal fibroblasts. However, the amount of the alpha chain precursor is greatly reduced. Mature chains were not detected. The one healthy adult we studied displayed a nonuniform distribution of Hex A; while it lacked activity in the serum it had intermediate activity in fibroblasts and leukocytes.

Elevated serum CK-BB levels in patients with cerebral transtentorial herniation after ischemic stroke.

We examined the time course of CK and its isoenzymes in 15 patients with severe ischemic stroke. Patients with cerebral transtentorial herniation (n = 7) had the highest CK-BB activity during herniation (1.54 +/- 0.6 U/L, mean +/- SD; range: 1.0-2.6 U/L). These values were distinctly above the values of a control group of 20 patients with non-neurological diseases (0.39 +/- 0.2 U/L, mean +/- SD). In patients with smaller lesions without herniation (n = 8) the maximum CK-BB increase was lower (0.56 +/- 0.26 U/L, mean +/- SD).

Radioimmunoassay of brain-type creatine kinase-BB isoenzyme in human tissues and in serum of patients with neurological disorders.

A sensitive radioimmunoassay for human brain type creatine kinase-BB isoenzyme has been used to measure the protein in serum as a potential index of central nervous system damage in patients with neurological disorders. The MB form of the enzyme (but not the MM form) partially cross-reacts in the radioimmunoassay but shows non-parallel dilution characteristics which distinguish it from the BB form of the enzyme. A survey of human tissues shows immunoreactive creatine kinase-BB in all human organs, with intestine and prostate approaching 35% of the brain concentration in contrast to the particularly low levels found in skeletal muscle, liver, spleen, and erythrocytes. No serum factors interfering in the assay were detected, and the normal serum level of creatine kinase-BB was found to be approximately 1-3 ng/ml. Serum containing high levels of immunoreactivity showed dilution characteristics paralleling the standard curve in the radioimmunoassay. Measurements of the protein in 47 patients with a variety of neurological disorders showed significantly raised mean levels in patients with dementia and with head injuries, isolated raised levels were also found in other disorders.

An investigation of pyruvate metabolism in patients with cerebellar and spinocerebellar degeneration.

This study extends previous observations of pyruvate metabolism in the spino-cerebellar degenerations by screening for abnormalities of pyruvate oxidation using the rise in blood pyruvate after an oral glucose load and examining the activity of the lipoamide dehydrogenase (LAD) moeity of the pyruvate dehydrogenase complex in the serum of 31 patients with Friedreich's ataxia, hereditary spastic ataxia and primary cerebellar degeneration. Serum LAD activity was significantly reduced in 10 Friedreich's ataxia patients when compared to controls and to 10 patients with spastic ataxia, thus confirming previous studies. Two patients with Friedreich's ataxia and 2 with primary cerebellar degeneration had abnormal blood pyruvate curves after oral glucose loading. The findings suggest that abnormal pyruvate oxidation occurs in some cases of Friedreich's ataxia and primary cerebellar degeneration and that the abnormality of pyruvate metabolism is not necessarily reflected in the serum LAD activity of these patients. The relevance of these findings to the heterogeneity of the hereditary ataxias is discussed.