7T MRI detects widespread brain iron deposition in neuroferritinopathy.

Neuroferritinopathy is a disorder of neurodegeneration with brain iron accumulation that has no proven disease-modifying treatments. Clinical trials require biomarkers of iron deposition. We examined brain iron accumulation in one presymptomatic FTL mutation carrier, two individuals with neuroferritinopathy and one healthy control using ultra-high-field 7T MRI. There was increased magnetic susceptibility, suggestive of iron deposition, in superficial and deep gray matter in both presymptomatic and symptomatic neuroferritinopathy. Cavitation of the putamen and globus pallidus increased with disease stage and at follow up. The widespread brain iron deposition in presymptomatic and early disease provides an opportunity for monitoring disease-modifying intervention.

Exploring therapeutic strategies for infantile neuronal axonal dystrophy (INAD/PARK14).

Infantile neuroaxonal dystrophy (INAD) is caused by recessive variants in PLA2G6 and is a lethal pediatric neurodegenerative disorder. Loss of the Drosophila homolog of PLA2G6, leads to ceramide accumulation, lysosome expansion, and mitochondrial defects. Here, we report that retromer function, ceramide metabolism, the endolysosomal pathway, and mitochondrial morphology are affected in INAD patient-derived neurons. We show that in INAD mouse models, the same features are affected in Purkinje cells, arguing that the neuropathological mechanisms are evolutionary conserved and that these features can be used as biomarkers. We tested 20 drugs that target these pathways and found that Ambroxol, Desipramine, Azoramide, and Genistein alleviate neurodegenerative phenotypes in INAD flies and INAD patient-derived neural progenitor cells. We also develop an AAV-based gene therapy approach that delays neurodegeneration and prolongs lifespan in an INAD mouse model.

Mouse models of NADK2 deficiency analyzed for metabolic and gene expression changes to elucidate pathophysiology.

NADK2 encodes the mitochondrial form of nicotinamide adenine dinucleotide (NAD) kinase, which phosphorylates NAD. Rare recessive mutations in human NADK2 are associated with a syndromic neurological mitochondrial disease that includes metabolic changes, such as hyperlysinemia and 2,4 dienoyl CoA reductase (DECR) deficiency. However, the full pathophysiology resulting from NADK2 deficiency is not known. Here, we describe two chemically induced mouse mutations in Nadk2-S326L and S330P-which cause severe neuromuscular disease and shorten lifespan. The S330P allele was characterized in detail and shown to have marked denervation of neuromuscular junctions by 5 weeks of age and muscle atrophy by 11 weeks of age. Cerebellar Purkinje cells also showed progressive degeneration in this model. Transcriptome profiling on brain and muscle was performed at early and late disease stages. In addition, metabolomic profiling was performed on the brain, muscle, liver and spinal cord at the same ages and on plasma at 5 weeks. Combined transcriptomic and metabolomic analyses identified hyperlysinemia, DECR deficiency and generalized metabolic dysfunction in Nadk2 mutant mice, indicating relevance to the human disease. We compared findings from the Nadk model to equivalent RNA sequencing and metabolomic datasets from a mouse model of infantile neuroaxonal dystrophy, caused by recessive mutations in Pla2g6. This enabled us to identify disrupted biological processes that are common between these mouse models of neurological disease, as well as those processes that are gene-specific. These findings improve our understanding of the pathophysiology of neuromuscular diseases and describe mouse models that will be useful for future preclinical studies.

Genome sequencing reveals novel noncoding variants in PLA2G6 and LMNB1 causing progressive neurologic disease.

Neurodegenerative disorders and leukodystrophies are progressive neurologic conditions that can occur following the disruption of intricately coordinated patterns of gene expression. Exome sequencing has been adopted as an effective diagnostic tool for determining the underlying genetic etiology of Mendelian neurologic disorders, however genome sequencing offer advantages in its ability to identify and characterize copy number, structural, and sequence variants in noncoding regions. Genome sequencing from peripheral leukocytes was performed on two patients with progressive neurologic disease of unknown etiology following negative genetic investigations including exome sequencing. RNA sequencing from peripheral blood was performed to determine gene expression patterns in one of the patients. Potential causative variants were matched to the patients' clinical presentation. The first proband was found to be heterozygous for a likely pathogenic missense variant in PLA2G6 (c.386T>C; p.Leu129Pro) and have an additional deep intronic variant in PLA2G6 (c.2035-926G>A). RNA sequencing indicated this latter variant created a splice acceptor site leading to the incorporation of a pseudo-exon introducing a premature termination codon. The second proband was heterozygous for a 261 kb deletion upstream of LMNB1 that included an enhancer region. Previous reports of copy number variants spanning this region of cis-acting regulatory elements corroborated its pathogenicity. When combined with clinical presentations, these findings led to a definitive diagnosis of autosomal recessive infantile neuroaxonal dystrophy and autosomal dominant adult-onset demyelinating leukodystrophy, respectively. In patients with progressive neurologic disease of unknown etiology, genome sequencing with the addition of RNA analysis where appropriate should be considered for the identification of causative noncoding pathogenic variants.

Interaction between TRPML1 and p62 in Regulating Autophagosome-Lysosome Fusion and Impeding Neuroaxonal Dystrophy in Alzheimer's Disease.

The loss of transient receptor potential mucolipin 1 (TRPML1), an endosomal and lysosomal Ca2+-releasing channel, has been implicated in neurodegenerative disorders. Mounting evidence have shown that TRPML1 could clear intraneuronal amyloid-ß (Aß), which triggers a hypothesis that TRPML1 activation may be beneficial for axonal transport in Alzheimer's disease (AD). In this work, the functional roles of TRPML1 were studied in the APP/PS1 transgenic mice and Aß1-42-stimulated hippocampal neurons HT22. We found that lentivirus-mediated overexpression of TRPML1 was shown to promote an accumulation of autolysosomes and increase brain-derived neurotrophic factor (BDNF) transportation to the nucleus, suggesting an axon-protective function. More importantly, we found that TRPML1 also increased p62 that interacted with dynein. Lentivirus-mediated knockdown of p62 or inhibition of dynein by ciliobrevin D stimulation was found to reduce autolysosome formation and nuclear accumulation of BDNF in HT22 cells with Aß1-42 stimulation. Inhibition of p62 by XRK3F2 stimulation was observed to promote the death of hippocampal neurons of the APP/PS1 transgenic mice. TRPML1 recruited dynein by interacting with p62 to promote the autophagosome-lysosome fusion to mediate BDNF nuclear translocation to impede axon dystrophy in mice with Alzheimer-like phenotypes. In summary, these results demonstrate the presence of a TRPML1/p62/dynein regulatory network in AD, and activation of TRPML1 is required for axon protection to prevent neuroaxonal dystrophy.

Vitamin E prevents lipid peroxidation and iron accumulation in PLA2G6-Associated Neurodegeneration.

BACKGROUND: PLA2G6-Associated Neurodegeneration (PLAN) is a rare neurodegenerative disease with autosomal recessive inheritance, which belongs to the NBIA (Neurodegeneration with Brain Iron Accumulation) group. Although the pathogenesis of the disease remains largely unclear, lipid peroxidation seems to play a central role in the pathogenesis. Currently, there is no cure for the disease. OBJECTIVE: In this work, we examined the presence of lipid peroxidation, iron accumulation and mitochondrial dysfunction in two cellular models of PLAN, patients-derived fibroblasts and induced neurons, and assessed the effects of α-tocopherol (vitamin E) in correcting the pathophysiological alterations in PLAN cell cultures. METHODS: Pathophysiological alterations were examined in fibroblasts and induced neurons generated by direct reprograming. Iron and lipofuscin accumulation were assessed using light and electron microscopy, as well as biochemical analysis techniques. Reactive Oxygen species production, lipid peroxidation and mitochondrial dysfunction were measured using specific fluorescent probes analysed by fluorescence microscopy and flow cytometry. RESULTS: PLAN fibroblasts and induced neurons clearly showed increased lipid peroxidation, iron accumulation and altered mitochondrial membrane potential. All these pathological features were reverted with vitamin E treatment. CONCLUSIONS: PLAN fibroblasts and induced neurons reproduce the main pathological alterations of the disease and provide useful tools for disease modelling. The main pathological alterations were corrected by Vitamin E supplementation in both models, suggesting that blocking lipid peroxidation progression is a critical therapeutic target.

Physiological significance of WDR45, a responsible gene for ß-propeller protein associated neurodegeneration (BPAN), in brain development.

WDR45 plays an essential role in the early stage of autophagy. De novo heterozygous mutations in WDR45 have been known to cause ß-propeller protein-associated neurodegeneration (BPAN), a subtype of neurodegeneration with brain iron accumulation (NBIA). Although BPAN patients display global developmental delay with intellectual disability, the neurodevelopmental pathophysiology of BPAN remains largely unknown. In the present study, we analyzed the physiological role of Wdr45 and pathophysiological significance of the gene abnormality during mouse brain development. Morphological and biochemical analyses revealed that Wdr45 is expressed in a developmental stage-dependent manner in mouse brain. Wdr45 was also found to be located in excitatory synapses by biochemical fractionation. Since WDR45 mutations are thought to cause protein degradation, we conducted acute knockdown experiments by in utero electroporation in mice to recapitulate the pathophysiological conditions of BPAN. Knockdown of Wdr45 caused abnormal dendritic development and synaptogenesis during corticogenesis, both of which were significantly rescued by co-expression with RNAi-resistant version of Wdr45. In addition, terminal arbors of callosal axons were less developed in Wdr45-deficient cortical neurons of adult mouse when compared to control cells. These results strongly suggest a pathophysiological significance of WDR45 gene abnormalities in neurodevelopmental aspects of BPAN.

Pathogenic mechanism and modeling of neuroferritinopathy.

Neuroferritinopathy is a rare autosomal dominant inherited movement disorder caused by alteration of the L-ferritin gene that results in the production of a ferritin molecule that is unable to properly manage iron, leading to the presence of free redox-active iron in the cytosol. This form of iron has detrimental effects on cells, particularly severe for neuronal cells, which are highly sensitive to oxidative stress. Although very rare, the disorder is notable for two reasons. First, neuroferritinopathy displays features also found in a larger group of disorders named Neurodegeneration with Brain Iron Accumulation (NBIA), such as iron deposition in the basal ganglia and extrapyramidal symptoms; thus, the elucidation of its pathogenic mechanism may contribute to clarifying the incompletely understood aspects of NBIA. Second, neuroferritinopathy shows the characteristic signs of an accelerated process of aging; thus, it can be considered an interesting model to study the progress of aging. Here, we will review the clinical and neurological features of neuroferritinopathy and summarize biochemical studies and data from cellular and animal models to propose a pathogenic mechanism of the disorder.

Neuropathological and biochemical investigation of Hereditary Ferritinopathy cases with ferritin light chain mutation: Prominent protein aggregation in the absence of major mitochondrial or oxidative stress.

AIMS: Neuroferritinopathy (NF) or hereditary ferritinopathy (HF) is an autosomal dominant movement disorder due to mutation in the light chain of the iron storage protein ferritin (FTL). HF is the only late-onset neurodegeneration with brain iron accumulation disorder and study of HF offers a unique opportunity to understand the role of iron in more common neurodegenerative syndromes. METHODS: We carried out pathological and biochemical studies of six individuals with the same pathogenic FTL mutation. RESULTS: CNS pathological changes were most prominent in the basal ganglia and cerebellar dentate, echoing the normal pattern of brain iron accumulation. Accumulation of ferritin and iron was conspicuous in cells with a phenotype suggesting oligodendrocytes, with accompanying neuronal pathology and neuronal loss. Neurons still survived, however, despite extensive adjacent glial iron deposition, suggesting neuronal loss is a downstream event. Typical age-related neurodegenerative pathology was not normally present. Uniquely, the extensive aggregates of ubiquitinated ferritin identified indicate that abnormal FTL can aggregate, reflecting the intrinsic ability of FTL to self-assemble. Ferritin aggregates were seen in neuronal and glial nuclei showing parallels with Huntington's disease. There was neither evidence of oxidative stress activation nor any significant mitochondrial pathology in the affected basal ganglia. CONCLUSIONS: HF shows hallmarks of a protein aggregation disorder, in addition to iron accumulation. Degeneration in HF is not accompanied by age-related neurodegenerative pathology and the lack of evidence of oxidative stress and mitochondrial damage suggests that these are not key mediators of neurodegeneration in HF, casting light on other neurodegenerative diseases characterized by iron deposition.

Compound heterozygous PLA2G6 loss-of-function variants in Swaledale sheep with neuroaxonal dystrophy.

Sporadic occurrences of neurodegenerative disorders including neuroaxonal dystrophy (NAD) have been previously reported in sheep. However, so far no causative genetic variant has been found for ovine NAD. The aim of this study was to characterize the phenotype and the genetic aetiology of an early-onset neurodegenerative disorder observed in several lambs of purebred Swaledale sheep, a native English breed. Affected lambs showed progressive ataxia and stiff gait and subsequent histopathological analysis revealed the widespread presence of axonal spheroid indicating neuronal degeneration. Thus, the observed clinical phenotype could be explained by a novel form of NAD. After SNP genotyping and subsequent linkage mapping within a paternal half-sib pedigree with a total of five NAD-affected lambs, we identified two loss-of-function variants by whole-genome sequencing in the ovine PLA2G6 gene situated in a NAD-linked genome region on chromosome 3. All cases were carriers of a compound heterozygous splice site variant in intron 2 and a nonsense variant in exon 8. Herein we present evidence for the occurrence of a familial novel form of recessively inherited NAD in sheep due to allelic heterogeneity at PLA2G6. This study reports two pathogenic variants in PLA2G6 causing a novel form of NAD in Swaledale sheep which enables selection against this fatal disorder.

Cryo-EM structures and functional characterization of homo- and heteropolymers of human ferritin variants.

The role of abnormal brain iron metabolism in neurodegenerative diseases is still insufficiently understood. Here, we investigate the molecular basis of the neurodegenerative disease hereditary ferritinopathy (HF), in which dysregulation of brain iron homeostasis is the primary cause of neurodegeneration. We mutagenized ferritin's three-fold pores (3FPs), i.e. the main entry route for iron, to investigate ferritin's iron management when iron must traverse the protein shell through the disrupted four-fold pores (4FPs) generated by mutations in the ferritin light chain (FtL) gene in HF. We assessed the structure and properties of ferritins using cryo-electron microscopy and a range of functional analyses in vitro. Loss of 3FP function did not alter ferritin structure but led to a decrease in protein solubility and iron storage. Abnormal 4FPs acted as alternate routes for iron entry and exit in the absence of functional 3FPs, further reducing ferritin iron-storage capacity. Importantly, even a small number of MtFtL subunits significantly compromises ferritin solubility and function, providing a rationale for the presence of ferritin aggregates in cell types expressing different levels of FtLs in patients with HF. These findings led us to discuss whether modifying pores could be used as a pharmacological target in HF.

Loss of tyrosine hydroxylase, motor deficits and elevated iron in a mouse model of phospholipase A2G6-associated neurodegeneration (PLAN).

Phospholipase A2G6-associated neurodegeneration (PLAN) is a rare early-onset monogenic neurodegenerative movement disorder which targets the basal ganglia and other regions in the central and peripheral nervous system; presenting as a series of heterogenous subtypes in patients. We describe here a B6.C3-Pla2g6m1J/CxRwb mouse model of PLAN which presents with early-onset neurodegeneration at 90 days which is analogous of the disease progression that is observed in PLAN patients. Homozygous mice had a progressively worsening motor deficit, which presented as tremors starting at 65 days and progressed to severe motor dysfunction and increased falls on the wire hang test at 90 days. This motor deficit positively correlated with a reduction in tyrosine hydroxylase (TH) protein expression in dopaminergic neurons of the substantia nigra (SN) without any neuronal loss. Fluorescence imaging of Thy1-YFP revealed spheroid formation in the SN. The spheroids in homozygous mice strongly mirrors those observed in patients and were demonstrated to correlate strongly with the motor deficits as measured by the wire hang test. The appearance of spheroids preceded TH loss and increased spheroid numbers negatively correlated with TH expression. Perls/DAB staining revealed the presence of iron accumulation within the SN of mice. This mouse model captures many of the major hallmarks of PLAN including severe-early onset neurodegeneration, a motor deficit that correlates directly to TH levels, spheroid formation and iron accumulation within the basal ganglia. Thus, this mouse line is a useful tool for further research efforts to improve understanding of how these disease mechanisms give rise to the disease presentations seen in PLAN patients as well as to test novel therapies.

Stem Cell Modeling of Neuroferritinopathy Reveals Iron as a Determinant of Senescence and Ferroptosis during Neuronal Aging.

Neuroferritinopathy (NF) is a movement disorder caused by alterations in the L-ferritin gene that generate cytosolic free iron. NF is a unique pathophysiological model for determining the direct consequences of cell iron dysregulation. We established lines of induced pluripotent stem cells from fibroblasts from two NF patients and one isogenic control obtained by CRISPR/Cas9 technology. NF fibroblasts, neural progenitors, and neurons exhibited the presence of increased cytosolic iron, which was also detectable as: ferritin aggregates, alterations in the iron parameters, oxidative damage, and the onset of a senescence phenotype, particularly severe in the neurons. In this spontaneous senescence model, NF cells had impaired survival and died by ferroptosis. Thus, non-ferritin-bound iron is sufficient per se to cause both cell senescence and ferroptotic cell death in human fibroblasts and neurons. These results provide strong evidence supporting the primary role of iron in neuronal aging and degeneration.

Mutant L-chain ferritins that cause neuroferritinopathy alter ferritin functionality and iron permeability.

In mammals, the iron storage and detoxification protein ferritin is composed of two functionally and genetically distinct subunit types, H (heavy) and L (light). The two subunits co-assemble in various ratios, with a tissue specific distribution, to form shell-like protein structures of 24 subunits within which a mineralized iron core is stored. The H-subunits possess ferroxidase centers that catalyze the rapid oxidation of ferrous ions, whereas the L-subunit does not have such centers and is believed to play an important role in electron transfer reactions that occur during the uptake and release of iron. Pathogenic mutations on the L-chain lead to neuroferritinopathy, a neurodegenerative disease characterized by abnormal accumulation of ferritin inclusion bodies and iron in the central nervous system. Here, we have characterized the thermal stability, iron loading capacity, iron uptake, and iron release properties of ferritin heteropolymers carrying the three pathogenic L-ferritin mutants (L154fs, L167fs, and L148fs, which for simplicity we named Ln1, Ln2 and Ln3, respectively), and a non-pathogenic variant (L135P) bearing a single substitution on the 3-fold axes of L-subunits. The UV-Vis data show a similar iron loading capacity (ranging between 1800 to 2400 Fe(iii)/shell) for all ferritin samples examined in this study, with Ln2 holding the least amount of iron (i.e. 1800 Fe(iii)/shell). The three pathogenic L-ferritin mutants revealed higher rates of iron oxidation and iron release, suggesting that a few mutated L-chains on the heteropolymer have a significant effect on iron permeability through the ferritin shell. DSC thermograms showed a strong destabilization effect, the severity of which depends on the location of the frameshift mutations (i.e. wt heteropolymer ferritin ≅ homopolymer H-chain > L135P > Ln2 > Ln1 > Ln3). Variant L135P had only minor effects on the protein functionality and stability, suggesting that local melting of the 3-fold axes in this variant may not be responsible for neuroferritinopathy-like disorders. The data support the hypothesis that hereditary neuroferritinopathies are due to alterations of ferritin functionality and lower physical stability which correlate with the frameshifts introduced at the C-terminal sequence and explain the dominant transmission of the disorder.

Neurodegeneration with brain iron accumulation: Insights into the mitochondria dysregulation.

NBIA (Neurodegeneration with brain iron accumulation) is a group of inherited neurologic disorders characterized by marked genetic heterogeneity, in which iron atypical accumulates in basal ganglia resulting in brain magnetic resonance imaging changes, histopathological abnormalities, and neuropsychiatric clinical symptoms. With the rapid development of high-throughput sequencing technologies, ten candidate genes have been identified, including PANK2, PLA2G6, C19orf12, WDR45, FA2H, ATP13A2, FTL, CP, C2orf37, and COASY. They are involved in seemingly unrelated cellular pathways, such as iron homeostasis (FTL, CP), lipid metabolism (PLA2G6, C19orf12, FA2H), Coenzyme A synthesis (PANK2, COASY), and autophagy (WDR45, ATP13A2). In particular, PANK2, COASY, PLA2G6, and C19orf12 are located on mitochondria, which associate with certain subtypes of NBIA showing mitochondria dysregulation. However, the relationships among those four genes are still unclear. Therefore, this review is specifically focused on dysregulation of mitochondria in NBIA and afore-mentioned four genes, with summaries of both pathological and clinical findings.

Autosomal dominant mitochondrial membrane protein-associated neurodegeneration (MPAN).

BACKGROUND: Mitochondrial membrane protein-associated neurodegeneration (MPAN) is caused by pathogenic sequence variants in C19orf12. Autosomal recessive inheritance has been demonstrated. We present evidence of autosomal dominant MPAN and propose a mechanism to explain these cases. METHODS: Two large families with apparently dominant MPAN were investigated; additional singleton cases of MPAN were identified. Gene sequencing and multiplex ligation-dependent probe amplification were used to characterize the causative sequence variants in C19orf12. Post-mortem brain from affected subjects was examined. RESULTS: In two multi-generation non-consanguineous families, we identified different nonsense sequence variations in C19orf12 that segregate with the MPAN phenotype. Brain pathology was similar to that of autosomal recessive MPAN. We additionally identified a preponderance of cases with single heterozygous pathogenic sequence variants, including two with de novo changes. CONCLUSIONS: We present three lines of clinical evidence to demonstrate that MPAN can manifest as a result of only one pathogenic C19orf12 sequence variant. We propose that truncated C19orf12 proteins, resulting from nonsense variants in the final exon in our autosomal dominant cohort, impair function of the normal protein produced from the non-mutated allele via a dominant negative mechanism and cause loss of function. These findings impact the clinical diagnostic evaluation and counseling.

Structure, Function, Folding, and Aggregation of a Neuroferritinopathy-Related Ferritin Variant.

Neuroferritinopathy is a rare, adult-onset, dominantly inherited movement disorder caused by mutations in the ferritin gene. A ferritin light-chain variant related to neuroferritinopathy, in which alanine 96 is replaced with threonine (A96T), was expressed in Escherichia coli, purified, and characterized. The circular dichroism, analytical ultracentrifugation, and small-angle X-ray scattering studies have shown that both the subunit structure and the assembly of A96T are the same as those of wild-type human ferritin light chain (HuFTL). The iron-incorporation ability was also comparable to that of HuFTL. Although the structural stability against heat, acid, and denaturant was reduced, the structure was sufficiently stable under physiological conditions. The most remarkable defects observed for A96T were a lower refolding efficiency and a stronger propensity to aggregate. The possible relationship between folding deficiency and disease is discussed.

Impaired iPLA2ß activity affects iron uptake and storage without iron accumulation: An in vitro study excluding decreased iPLA2ß activity as the cause of iron deposition in PLAN.

PLA2G6-associated neurodegeneration (PLAN, NBIA2) is the second most common type of neurodegeneration with brain iron accumulation (NBIA), caused by recessive mutations of PLA2G6 gene, which encodes Ca2+-independent phospholipase A2ß (iPLA2ß). In most PLAN cases, decreased iPLA2ß activity and iron deposition was observed meanwhile, and researchers also identified a PLA2G6 mutation family without iron deposition shown by MRI images. This brought us the question of whether decreased iPLA2ß activity was the cause of iron deposition in PLAN. In this study, we used S-BEL as the antagonist of iPLA2ß to block its activity and used SH-SY5Y cells as the expression system. We incubated SH-SY5Y cells with different concentrations of S-BEL. The results showed that decreased iPLA2ß activity led no obvious iron accumulation, while changes of cells state and activation of apoptosis were observed. To further investigate the cause of unchanged iron level, we examined the cellular iron regulatory proteins involved in iron uptake, storage and export. The results were as follows: TfR1 (iron uptake protein) expression was decreased, the expression of ferritin heavy chain and light chain (iron storage protein) was increased. There was no alteration of the expression of DMT1 (iron uptake protein) and FPN1 (iron export protein). Under the condition of decreased iPLA2ß activity, there was no obvious iron accumulation but iron uptake activity decreased and iron storage activity increased. Therefore, we speculate that the decreased iPLA2ß activity may not be the main reason for iron deposition in PLAN.