Protective Effect of Pemafibrate Treatment against Diabetic Retinopathy in Spontaneously Diabetic Torii Fatty Rats.

Diabetic retinopathy (DR) can cause visual impairment and blindness, and the increasing global prevalence of diabetes underscores the need for effective therapies to prevent and treat DR. Therefore, this study aimed to evaluate the protective effect of pemafibrate treatment against DR, using a Spontaneously Diabetic Torii (SDT) fatty rat model of obese type 2 diabetes. SDT fatty rats were fed either a diet supplemented with pemafibrate (0.3 mg/kg/d) for 16 weeks, starting at 8 weeks of age (Pf SDT fatty: study group), or normal chow (SDT fatty: controls). Normal chow was provided to Sprague-Dawley (SD) rats (SD: normal controls). Electroretinography (ERG) was performed at 8 and 24 weeks of age to evaluate the retinal neural function. After sacrifice, retinal thickness, number of retinal folds, and choroidal thickness were evaluated, and immunostaining was performed for aquaporin-4 (AQP4). No significant differences were noted in food consumption, body weight, or blood glucose level after pemafibrate administration. Triglyceride levels were reduced, and high-density lipoprotein cholesterol levels were increased. Extension of oscillatory potential (OP)1 and OP3 waves on ERG was suppressed in the Pf SDT fatty group. Retinal thickness at 1500 microns from the optic disc improved in the Pf SDT fatty group. No significant improvements were noted in choroidal thickness or number of retinal folds. Quantitative analyses showed that AQP4-positive regions in the retinas were significantly larger in the Pf SDT fatty group than in the SDT fatty group. The findings suggest that pemafibrate treatment can exert protective effects against DR.

The Role of GABA in the Dorsal Striatum-Raphe Nucleus Circuit Regulating Stress Vulnerability in Male Mice with High Levels of Shati/Nat8l.

Depression is a frequent and serious illness, and stress is considered the main risk factor for its onset. First-line antidepressants increase serotonin (5-hydroxytryptamine; 5-HT) levels in the brain. We previously reported that an N-acetyltransferase, Shati/Nat8l, is upregulated in the dorsal striatum (dSTR) of stress-susceptible mice exposed to repeated social defeat stress (RSDS) and that dSTR Shati/Nat8l overexpression in mice (dSTR-Shati OE) induces stress vulnerability and local reduction in 5-HT content. Male mice were used in this study, and we found that dSTR 5-HT content decreased in stress-susceptible but not in resilient mice. Moreover, vulnerability to stress in dSTR-Shati OE mice was suppressed by the activation of serotonergic neurons projecting from the dorsal raphe nucleus (dRN) to the dSTR, followed by upregulation of 5-HT content in the dSTR using designer receptors exclusively activated by designer drugs (DREADD). We evaluated the role of GABA in modulating the serotonergic system in the dRN. Stress-susceptible after RSDS and dSTR-Shati OE mice exhibited an increase in dRN GABA content. Furthermore, dRN GABA content was correlated with stress sensitivity. We found that the blockade of GABA signaling in the dRN suppressed stress susceptibility in dSTR-Shati OE mice. In conclusion, we propose that dSTR 5-HT and dRN GABA, controlled by striatal Shati/Nat8l via the dSTR-dRN neuronal circuitry, critically regulate stress sensitivity. Our study provides insights into the neural processes that underlie stress and suggests that dSTR Shati/Nat8l could be a novel therapeutic target for drugs against depression, allowing direct control of the dRN serotonergic system.

Efficient gene transduction in pigs and macaques with the engineered AAV vector AAV.GT5 for hemophilia B gene therapy.

Gene therapy using adeno-associated virus (AAV)-based vectors has become a realistic therapeutic option for hemophilia. We examined the potential of a novel engineered liver-tropic AAV3B-based vector, AAV.GT5, for hemophilia B gene therapy. In vitro transduction with AAV.GT5 in human hepatocytes was more than 100 times higher than with AAV-Spark100, another bioengineered vector used in a clinical trial. However, liver transduction following intravenous injection of these vectors was similar in mice with a humanized liver and in macaques. This discrepancy was due to the low recovery and short half-life of AAV.GT5 in blood, depending on the positive charge of the heparin-binding site in the capsid. Bypassing systemic clearance with the intra-hepatic vascular administration of AAV.GT5, but not AAV-Spark100, enhanced liver transduction in pigs and macaques. AAV.GT5 did not develop neutralizing antibodies (NAbs) in two of four animals, while AAV-Spark100 induced serotype-specific NAbs in all macaques tested (4 of 4). The NAbs produced after AAV-Spark100 administration were relatively serotype specific, and challenge with AAV.GT5 through the hepatic artery successfully boosted liver transduction in one animal previously administered AAV-Spark100. In summary, AAV.GT5 showed different vector kinetics and NAb induction compared with AAV-Spark100, and intra-hepatic vascular administration may minimize the vector dose required and vector dissemination.

Enhanced Cochlear Transduction by AAV9 with High-Concentration Sucrose.

The inner ear is a primary lesion in sensorineural hearing loss and has been a target in gene therapy. The efficacy of gene therapy depends on achieving sufficient levels of transduction at a safe vector dose. Vectors derived from various adeno-associated viruses (AAVs) are predominantly used to deliver therapeutic genes to inner ear cells. AAV9 and its variants vector are attractive candidates for clinical applications since they can cross the mesothelial cell layer and transduce inner hair cells (IHCs), although this requires relatively high doses. In this study, we investigated the effects of sucrose on the transduction of a variant of the AAV9 vector for gene transfer in the inner ear. We found that high concentrations of sucrose increased gene transduction in House Ear Institute-Organ of Corti 1 (HEI-OC1) cells in vitro. In addition, we demonstrated that simultaneous administration of sucrose enhanced the transduction of mouse IHCs and spiral ligament cells using an AAV9 variant vector. The procedure did not increase the thresholds in the auditory brainstem response, suggesting that sucrose had no adverse effect on auditory function. This versatile method may be valuable in the development of novel gene therapies for adult-onset sensorineural hearing loss.

[Kampo Medicine for Neurological Diseases].

Goreisan, Goshuyuto, Tokishakuyakusan, and Keishibukuryogan are frequently used to treat migraines. Goreisan is also used to treat chronic subdural hematoma. Yokukansan and Keishikaryukotsuboreito are useful for alleviating the behavioral and psychological symptoms of dementia. Keishikajyutsubuto and Shinbuto are used to treat numbness and pain associated with peripheral neuropathy. Hangeshashinto has been successfully attempted to treat intractable hiccoughs. It is advisable to use an extract of stable quality in accordance with the rule of thumb described in the classics. However, awareness of side effects, such as pseudoaldosteronism caused by licorice, is important.

In vivo analysis of aggregation propensity of low levels of mislocalized TDP-43 on cytopathological and behavioral phenotype of ALS/FTLD.

Mislocalization and aggregate formation of TAR DNA-biding protein of 43kD (TDP-43) in the cytoplasm are signatures of amyotrophic lateral sclerosis(ALS) and frontotemporal lobar degeneration (FTLD). However, the role of two cytopathologies in ALS/FTLD pathogenesis is unclear. This study aims to elucidate the difference in their causality of TDP-43 in ALS/FTLD in vivo, using transgenic mice expressing human TDP-43 with defective nuclear localizing signals in neurons (Cyto-TDP) and those with aggregation propensity (Cyto-aggTDP). The expression levels of both proteins are less than half of endogenous TDP-43. Despite the low amount of Cyto-aggTDP, the TDP-43 phosphorylation is more evident than Cyto-TDP. Histopathological study showed accelerated astrogliosis in the anterior cerebral cortex of both mice. Cyto-aggTDP mice demonstrated significant but faint loss of neurons in the perirhinal(PERI) and ectorhinal(ECT) areas and higher Iba1-staining in the spinal cord than aged control. Despite the lack of locomotor dysfunctions in both mice, the open-field test showed enhanced exploratory behavior, indicating that the perpetual mislocalization of TDP-43 may suffice to trigger FTLD behavior. Besides, the aggregation propensity of TDP-43 promotes phosphorylation, but its role in the clinicopathological phenotype may not be primary.

Overexpression of tyrosine hydroxylase in dopaminergic neurons increased sensitivity to methamphetamine.

Drug abuse is one of the great social problems in the world and a major healthcare challenge. It is supposed that sensitivity and reactivity to abuse drugs may vary from person to person, while its molecular basis is largely unknown. Dopaminergic neurons are deeply involved in addiction, and tyrosine hydroxylase (TH) catalyzes the first and rate-limiting step of the biosynthesis of dopamine (DA). We investigated the effects of increased TH expression on the metabolism of DA and reactivity to methamphetamine (METH), a drug of abuse, in mice. Wild-type TH (WT-TH) or the S40E mutant of TH (S40E-TH), which is an active form of TH mimicking phosphorylated TH at the 40th serine, was expressed in midbrain dopaminergic neurons using an adeno-associated virus (AAV) vector. The biochemical analysis showed that the turnover rates of DA in the nerve terminals were increased by the expression of WT-TH and S40E-TH, while there were few changes in the DA contents. Next, we administered METH to TH-overexpressing mice. We found that the S40E-TH-expressing mice responded to lower doses of METH than the control mice and WT-TH mice. The stereotyped behaviors appeared first in S40E-TH mice and then in WT-TH and control mice in this order. These data showed that the TH activity and expression level differentially affect DA metabolism in the nerve terminals from that in the cell bodies and that the TH activity and expression level are one of the determining factors for sensitivity and reactivity to METH. We suggest that TH may be a drug target for ameliorating sensitivity to drugs of abuse.

Early distribution of18 F-labeled AAV9 vectors in the cerebrospinal fluid after intracerebroventricular or intracisternal magna infusion in non-human primates.

BACKGROUND: The delivery of adeno-associated virus (AAV) vectors via the cerebrospinal fluid (CSF) has emerged as a valuable method for widespread transduction in the central nervous system. Although infusion into the cerebral ventricles is a common protocol in preclinical studies of small animals, the cisterna magna has been recognized as an alternative target for clinical studies because it can be reached in a less invasive manner using an intrathecal catheter via the subarachnoid space from a lumbar puncture. METHODS: We evaluated the early distribution of fluorine-18-labeled AAV9 vectors infused into the lateral ventricle or cisterna magna of four non-human primates using positron emission tomography. The expression of the green fluorescent protein was immunohistochemically determined. RESULTS: In both approaches, the labeled vectors diffused into the broad arachnoid space around the brain stem and cervical spinal cord within 30 min. Both infusion routes efficiently transduced neurons in the cervical spinal cord. CONCLUSIONS: For gene therapy that primarily targets the cervical spinal cord and brainstem, such as amyotrophic lateral sclerosis, cisterna magna infusion would be a feasible and effective administration method.

Adeno-associated virus vector-based gene therapies for pediatric diseases.

Gene therapy using adeno-associated virus (AAV) is a rapidly developing technology with widespread treatment potential. AAV2 vectors injected directly into the brain by stereotaxic brain surgery have shown good results in treating aromatic l-amino acid decarboxylase deficiency. Moreover, gene therapy using the AAV9 vector, which crosses the blood-brain barrier, has been performed in more than 2000 patients worldwide as a disease-modifying therapy for spinal muscular atrophy. AAV vectors have been applied to the development of gene therapies for various pediatric diseases. Gene therapy trials for hemophilia and ornithine transcarbamylase deficiency are underway. Clinical trials are planned for glucose transporter I deficiency, Niemann-Pick disease type C, and spinocerebellar ataxia type 1. The genome of AAV vectors is located in the episome and is rarely integrated into chromosomes, making the vectors safe. However, serious adverse events such as hepatic failure and thrombotic microangiopathy have been reported, and ongoing studies are focusing on developing more efficient vectors to reduce required dosages.

Hippocampus as a sorter and reverberatory integrator of sensory inputs.

The hippocampus must be capable of sorting and integrating multiple sensory inputs separately but simultaneously. However, it remains to be elucidated how the hippocampus executes these processes simultaneously during learning. Here we found that synchrony between conditioned stimulus (CS)-, unconditioned stimulus (US)- and future retrieval-responsible cells occurs in the CA1 during the reverberatory phase that emerges after sensory inputs have ceased, but not during CS and US inputs. Mutant mice lacking N-methyl-D-aspartate receptors (NRs) in CA3 showed a cued-fear memory impairment and a decrease in synchronized reverberatory activities between CS- and US-responsive CA1 cells. Optogenetic CA3 silencing at the reverberatory phase during learning impaired cued-fear memory. Thus, the hippocampus uses reverberatory activity to link CS and US inputs, and avoid crosstalk during sensory inputs.

Differences in the Effects of Pentobarbital Anesthetic and Combination of Medetomidine Hydrochloride, Midazolam, and Butorphanol Tartrate Anesthetic on Electroretinogram in Spontaneously Diabetic Torii Fatty Rats.

Purpose: The aim of this study was to investigate the effects of different anesthetic agents on electroretinograms (ERGs) in Spontaneously Diabetic Torii fatty rats (SDT fatty rats). Methods: The ERG recordings were measured under general anesthesia using pentobarbital or a combination of medetomidine hydrochloride, midazolam, and butorphanol (MMB) tartrate anesthesia in 12 9-week-old normal Sprague-Dawley rats (Jcl:SD rats) and 16 SDT fatty rats. Each animal model was divided into 2 groups, the pentobarbital group and MMB group. The amplitudes and peak times of the a- and b-waves and oscillatory potentials (OPs) were measured from 0.0001 candela per square meter (cd.s/m2) to 10.0 cd.s/m2. Results: The amplitude of the a-wave was significantly higher in the MMB group of Jcl:SD rats, but there was no significant difference in amplitude between the two groups of SDT fatty rats. There was no significant difference in the OP1 amplitude between both groups of Jcl:SD rats, but the OP1 amplitude was significantly higher in the MMB group of SDT fatty rats. The OP2 amplitude was significantly higher in the pentobarbital group in both the Jcl:SD rats and SDT fatty rats. There was no significant difference in the OP3 amplitude between the Jcl:SD and SDT fatty rat groups. The amplitude of the OP4 waves was significantly higher in the MMB group for both Jcl:SD and SDT fatty rats. There was no significant difference in the sums of the OP1 to OP4 (ΣOPs) amplitudes between the Jcl:SD and SDT fatty rat groups. There was no significant difference in the b-wave amplitude between the Jcl:SD rat groups, but the b-wave amplitude was significantly higher in the SDT fatty rats that received pentobarbital. The peak times for a-wave, OP1, OP2, OP3, OP4, and ΣOPs were significantly longer in the pentobarbital group of SD rats. The peak time of the b-wave was significantly longer in the MMB group of Jcl:SD rats, but the same result was obtained in the SDT fatty rats except that there was no significant difference in the a-wave. Conclusion: The overall ERG results vary depending on the anesthetic agent used. The OPs can be observed in detail when using MMB. Since the SDT fatty rat is a diabetic model animal, we recommend MMB as the anesthesia of choice when studying the OP waves in detail.

Conformational change of RNA-helicase DHX30 by ALS/FTD-linked FUS induces mitochondrial dysfunction and cytosolic aggregates.

Genetic mutations in fused in sarcoma (FUS) cause amyotrophic lateral sclerosis (ALS). Although mitochondrial dysfunction and stress granule have been crucially implicated in FUS proteinopathy, the molecular basis remains unclear. Here, we show that DHX30, a component of mitochondrial RNA granules required for mitochondrial ribosome assembly, interacts with FUS, and plays a crucial role in ALS-FUS. WT FUS did not affect mitochondrial localization of DHX30, but the mutant FUS lowered the signal of mitochondrial DHX30 and promoted the colocalization of cytosolic FUS aggregates and stress granule markers. The immunohistochemistry of the spinal cord from an ALS-FUS patient also confirmed the colocalization, and the immunoelectron microscope demonstrated decreased mitochondrial DHX30 signal in the spinal motor neurons. Subcellular fractionation by the detergent-solubility and density-gradient ultracentrifugation revealed that mutant FUS also promoted cytosolic mislocalization of DHX30 and aggregate formation. Interestingly, the mutant FUS disrupted the DHX30 conformation with aberrant disulfide formation, leading to impaired mitochondrial translation. Moreover, blue-native gel electrophoresis revealed an OXPHOS assembly defect caused by the FUS mutant, which was similar to that caused by DHX30 knockdown. Collectively, our study proposes DHX30 as a pivotal molecule in which disulfide-mediated conformational change mediates mitochondrial dysfunction and cytosolic aggregate formation in ALS-FUS.

Knockdown of Piccolo in the Nucleus Accumbens Suppresses Methamphetamine-Induced Hyperlocomotion and Conditioned Place Preference in Mice.

Methamphetamine (METH), the most widely distributed psychostimulant, aberrantly activates the reward system in the brain to induce addictive behaviors. The presynaptic protein "Piccolo", encoded by Pclo, was identified as a METH-responsive protein with enhanced expression in the nucleus accumbens (NAc) in mice. Although the physiological and pathological significance of Piccolo has been identified in dopaminergic signaling, its role in METH-induced behavioral abnormalities and the underlying mechanisms remain unclear. To clarify such functions, mice with Piccolo knockdown in the NAc (NAc-miPiccolo mice) by local injection of an adeno-associated virus vector carrying miRNA targeting Pclo were generated and investigated. NAc-miPiccolo mice exhibited suppressed hyperlocomotion, sensitization, and conditioned place preference behavior induced by systemic administration of METH. The excessive release of dopamine in the NAc was reduced in NAc-miPiccolo mice at baseline and in response to METH. These results suggest that Piccolo in the NAc is involved in METH-induced behavioral alterations and is a candidate therapeutic target for the treatment of drug addiction.

Tau-binding protein PRMT8 facilitates vacuole degeneration in the brain.

Amyloid-ß and tau pathologies are important factors leading to neurodegeneration in Alzheimer's disease (AD); however, the molecular mechanisms that link these pathologies remain unclear. Assuming that important though as yet unidentified factors inhibit/accelerate tau pathology and neuronal cell death under amyloid pathology, we sought to isolate and identify tau-interacting proteins from mouse brains with or without amyloid pathology. Among the proteins that were identified, we focused on protein arginine methyltransferase 8 (PRMT8), which interacts with tau specifically in the absence of amyloid pathology. To investigate the role of PRMT8 in the pathogenesis of AD, we conducted Prmt8 gene deletion and overexpression experiments in AppNL-G-F/MAPT double knock-in mice and analysed the resulting pathological alterations. PRMT8-knockout did not alter the AD pathology in double knock-in mice, whereas PRMT8-overexpression promoted tau phosphorylation, neuroinflammation and vacuole degeneration. To evaluate if such a PRMT8-induced vacuole degeneration depends on tau pathology, PRMT8 was overexpressed in tau-KO mice, which were consequently found to exhibit vacuole degeneration. In addition, proteomic analyses showed that PRMT8 overexpression facilitated the arginine methylation of vimentin. Abnormal protein methylation could be involved in PRMT8-induced brain pathologies. Taken together, PRMT8 may play an important role in the formation of tau pathology and vacuole degeneration.

Shati/Nat8l Overexpression Improves Cognitive Decline by Upregulating Neuronal Trophic Factor in Alzheimer's Disease Model Mice.

Alzheimer's disease (AD) is a type of dementia characterized by the deposition of amyloid ß, a causative protein of AD, in the brain. Shati/Nat8l, identified as a psychiatric disease related molecule, is a responsive enzyme of N-acetylaspartate (NAA) synthesis. In the hippocampi of AD patients and model mice, the NAA content and Shati/Nat8l expression were reported to be reduced. Having recently clarified the involvement of Shati/Nat8l in cognitive function, we examined the recovery effect of the hippocampal overexpression of Shati/Nat8l in AD model mice (5XFAD). Shati/Nat8l overexpression suppressed cognitive dysfunction without affecting the Aß burden or number of NeuN-positive neurons. In addition, brain-derived neurotrophic factor mRNA was upregulated by Shati/Nat8l overexpression in 5XFAD mice. These results suggest that Shati/Nat8l overexpression prevents cognitive dysfunction in 5XFAD mice, indicating that Shati/Nat8l could be a therapeutic target for AD.

Kampo Formula-Pattern Models: The Development of 13 New Clinically Useful Standard Abdominal Pattern Models in the Fukushin Simulator.

Aim: In Kampo medicine, there exists an important system of diagnosis called Fukushin, or abdominal diagnosis or palpation. By applying pressure to the abdomen of the patient, the physician can gain important information on the patient's physical state and use those indications to choose a suitable Kampo formulation. We have previously developed a Fukushin simulator, a teaching tool that reproduces the important abdominal patterns that doctors will encounter in clinical practice and that has received favourable feedback for students and practitioners. In order to make diagnosis and prescription easier, it is desirable to have matched formula-pattern pairings. The present study aims to develop such pairings. Methods: With the previously developed models as a foundation, in the present study the production team (two members) used materials such as urethane foam and silicone rubber to build an additional 13 standard abdominal pattern models matched to Kampo herbal formulas commonly used by practitioners in Japan. Subsequently, the evaluation team (the remaining 10 authors) investigated the viability of these models. Results: The evaluation team determined that abdominal pattern models matched to the following typical Kampo formulas were created successfully: Dai-saiko-To (), Dai-joki-To (), Shigyaku-San (), Saiko-ka-ryukotsu-borei-To (), Keishi-bukuryo-Gan (), Hachimi-jio-Gan (), Hange-shashin-To (), Sho-saiko-To (), Hochu-ekki-To (), Sho-kenchu-To (), Toki-shakuyaku-San (), Ninjin-To (), and Dai-kenchu-To (). Conclusion: We suggest that these new formula-pattern models can make an important contribution to the standardization of abdominal diagnosis and prescription and to Kampo education.

N-Acetyl Transferase, Shati/Nat8l, in the Dorsal Hippocampus Suppresses Aging-induced Impairment of Cognitive Function in Mice.

As the elderly population rapidly increases worldwide, the onset of cognitive dysfunction is expected to increase. Although neuronal plasticity, neurogenesis, and mitochondrial dysfunction have been reported to be involved in cognitive function, the detailed mechanism of cognitive impairment accompanied by aging is poorly understood as there are many confounding factors associated with aging. Therefore, effective treatments for aging have not yet been developed, and the establishment of therapeutic strategies has not progressed accordingly. We have previously found a decline of cognitive function in the developmental stage in mice who lack the expression of Shati/Nat8l, an N-acetyl transferase However, the contribution of Shati/Nat8l to cognitive impairment in aged mice has not yet been investigated. In this study, we aimed to investigate the role of Shati/Nat8l in cognitive function during aging. We observed a reduction in Shati/Nat8l mRNA expression in the dorsal hippocampus of mice as a result of their aging. Moreover, the cognitive dysfunction observed in aged mice was reversed by Shati/Nat8l overexpression in the dorsal hippocampus. Shati/Nat8l overexpression in the dorsal hippocampus of mice did not alter the expression of neurotrophic factors or mitochondrial function-related genes, including Bdnf or Pgc-1α, which are suggested to be downstream genes of Shati/Nat8l. Decreased N-acetyl aspartate (NAA) in aged mice was upregulated by Shati/Nat8l overexpression, suggesting that the Shati/Nat8l-NAA pathway determines cognitive function with aging. Taken together, Shati/Nat8l and NAA in the dorsal hippocampus may be novel targets for the treatment of cognitive impairment.

Retinoic acid receptor beta protects striatopallidal medium spiny neurons from mitochondrial dysfunction and neurodegeneration.

Retinoic acid is a powerful regulator of brain development, however its postnatal functions only start to be elucidated. We show that retinoic acid receptor beta (RARß), is involved in neuroprotection of striatopallidal medium spiny neurons (spMSNs), the cell type affected in different neuropsychiatric disorders and particularly prone to degenerate in Huntington disease (HD). Accordingly, the number of spMSNs was reduced in the striatum of adult Rarß-/- mice, which may result from mitochondrial dysfunction and neurodegeneration. Mitochondria morphology was abnormal in mutant mice whereas in cultured striatal Rarß-/- neurons mitochondria displayed exacerbated depolarization, and fragmentation followed by cell death in response to glutamate or thapsigargin-induced calcium increase. In vivo, Rarß-/- spMSNs were also more vulnerable to the mitochondrial toxin 3-nitropropionic acid (3NP), known to induce HD symptoms in human and rodents. In contrary, an RARß agonist, AC261066, decreased glutamate-induced toxicity in primary striatal neurons in vitro, and diminished mitochondrial dysfunction, spMSN cell death and motor deficits induced in wild type mice by 3NP. We demonstrate that the striatopallidal pathway is compromised in Rarß-/- mice and associated with HD-like motor abnormalities. Importantly, similar motor abnormalities and selective reduction of spMSNs were induced by striatal or spMSN-specific inactivation of RARß, further supporting a neuroprotective role of RARß in postnatal striatum.

WDR45 variants cause ferrous iron loss due to impaired ferritinophagy associated with nuclear receptor coactivator 4 and WD repeat domain phosphoinositide interacting protein 4 reduction.

Static encephalopathy of childhood with neurodegeneration in adulthood/ß-propeller protein-associated neurodegeneration is a neurodegenerative disorder with brain iron accumulation caused by the variants of WDR45, a core autophagy-related gene that encodes WD repeat domain phosphoinositide interacting protein 4. However, the pathophysiology of the disease, particularly the function of WDR45/WD repeat domain phosphoinositide interacting protein 4 in iron metabolism, is largely unknown. As no other variants of core autophagy-related genes show abnormalities in iron metabolism, the relation between autophagy and iron metabolism remains to be elucidated. Since iron deposition in the brain is the hallmark of static encephalopathy of childhood with neurodegeneration in adulthood/ß-propeller protein-associated neurodegeneration, iron chelation therapy has been attempted, but it was found to worsen the symptoms; thus, the establishment of a curative treatment is essential. Here, we evaluated autophagy and iron metabolism in patient-derived cells. The expression of ferritin and ferric iron increased and that of ferrous iron decreased in the patient cells with WDR45 variants. In addition, the expression of nuclear receptor coactivator 4 was markedly reduced in patient-derived cells. Furthermore, divalent metal transporter 1, which takes in ferrous iron, was upregulated, while ferroportin, which exports ferrous iron, was downregulated in patient-derived cells. The transfer of WDR45 via an adeno-associated virus vector restored WD repeat domain phosphoinositide interacting protein 4 and nuclear receptor coactivator 4 expression, reduced ferritin levels, and improved other phenotypes observed in patient-derived cells. As nuclear receptor coactivator 4 mediates the ferritin-specific autophagy, i.e. ferritinophagy, its deficiency impaired ferritinophagy, leading to the accumulation of ferric iron-containing ferritin and insufficiency of ferrous iron. Because ferrous iron is required for various essential biochemical reactions, the changes in divalent metal transporter 1 and ferroportin levels may indicate a compensatory response for maintaining the intracellular levels of ferrous iron. Our study revealed that the pathophysiology of static encephalopathy of childhood with neurodegeneration in adulthood/ß-propeller protein-associated neurodegeneration involves ferrous iron insufficiency via impaired ferritinophagy through nuclear receptor coactivator 4 expression reduction. Our findings could aid in developing a treatment strategy involving WDR45 manipulation, which may have clinical applications.