GID complex regulates the differentiation of neural stem cells by destabilizing TET2.

Brain development requires a delicate balance between self-renewal and differentiation in neural stem cells (NSC), which rely on the precise regulation of gene expression. Ten-eleven translocation 2 (TET2) modulates gene expression by the hydroxymethylation of 5-methylcytosine in DNA as an important epigenetic factor and participates in the neuronal differentiation. Yet, the regulation of TET2 in the process of neuronal differentiation remains unknown. Here, the protein level of TET2 was reduced by the ubiquitin-proteasome pathway during NSC differentiation, in contrast to mRNA level. We identified that TET2 physically interacts with the core subunits of the glucose-induced degradation-deficient (GID) ubiquitin ligase complex, an evolutionarily conserved ubiquitin ligase complex and is ubiquitinated by itself. The protein levels of GID complex subunits increased reciprocally with TET2 level upon NSC differentiation. The silencing of the core subunits of the GID complex, including WDR26 and ARMC8, attenuated the ubiquitination and degradation of TET2, increased the global 5-hydroxymethylcytosine levels, and promoted the differentiation of the NSC. TET2 level increased in the brain of the Wdr26+/- mice. Our results illustrated that the GID complex negatively regulates TET2 protein stability, further modulates NSC differentiation, and represents a novel regulatory mechanism involved in brain development.

Tet2 acts in the lateral habenula to regulate social preference in mice.

The lateral habenula (LHb) has been considered a moderator of social behaviors. However, it remains unknown how LHb regulates social interaction. Here, we show that the hydroxymethylase Tet2 is highly expressed in the LHb. Tet2 conditional knockout (cKO) mice exhibit impaired social preference; however, replenishing Tet2 in the LHb rescues social preference impairment in Tet2 cKO mice. Tet2 cKO alters DNA hydroxymethylation (5hmC) modifications in genes that are related to neuronal functions, as is confirmed by miniature two-photon microscopy data. Further, Tet2 knockdown in the glutamatergic neurons of LHb causes impaired social behaviors, but the inhibition of glutamatergic excitability restores social preference. Mechanistically, we identify that Tet2 deficiency reduces 5hmC modifications on the Sh3rf2 promoter and Sh3rf2 mRNA expression. Interestingly, Sh3rf2 overexpression in the LHb rescues social preference in Tet2 cKO mice. Therefore, Tet2 in the LHb may be a potential therapeutic target for social behavior deficit-related disorders such as autism.

5hmC modification regulates R-loop accumulation in response to stress.

R-loop, an RNA-DNA hybrid structure, arises as a transcriptional by-product and has been implicated in DNA damage and genomic instability when excessive R-loop is accumulated. Although previous study demonstrated that R-loop is associated with ten-eleven translocation (Tet) proteins, which oxidize 5-methylcytosine to 5-hydroxymethylcytosine (5hmC), the sixth base of DNA. However, the relationship between R-loop and DNA 5hmC modification remains unclear. In this study, we found that chronic restraint stress increased R-loop accumulation and decreased 5hmC modification in the prefrontal cortex (PFC) of the stressed mice. The increase of DNA 5hmC modification by vitamin C was accompanied with the decrease of R-loop levels; on the contrary, the decrease of DNA 5hmC modification by a small compound SC-1 increased the R-loop levels, indicating that 5hmC modification inversely regulates R-loop accumulation. Further, we showed that Tet deficiency-induced reduction of DNA 5hmC promoted R-loop accumulation. In addition, Tet proteins immunoprecipitated with Non-POU domain-containing octamer-binding (NONO) proteins. The deficiency of Tet proteins or NONO increased R-loop levels, but silencing Tet proteins and NONO did not further increase the increase accumulation, suggesting that NONO and Tet proteins formed a complex to inhibit R-loop formation. It was worth noting that NONO protein levels decreased in the PFC of stressed mice with R-loop accumulation. The administration of antidepressant fluoxetine to stressed mice increased NONO protein levels, and effectively decreased R-loop accumulation and DNA damage. In conclusion, we showed that DNA 5hmC modification negatively regulates R-loop accumulation by the NONO-Tet complex under stress. Our findings provide potential therapeutic targets for depression.

The Tet2-Upf1 complex modulates mRNA stability under stress conditions.

Introduction: Environmental stress promotes epigenetic alterations that impact gene expression and subsequently participate in the pathological processes of the disorder. Among epigenetic regulations, ten-eleven Translocation (Tet) enzymes oxidize 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) in DNA and RNA and function as critical players in the pathogenesis of diseases. Our previous results showed that chronic stress increases the expression of cytoplasmic Tet2 in the hippocampus of mice exposed to chronic mild stress (CMS). Whether the cytoplasmic Tet2 alters RNA 5hmC modification in chronic stress-related processes remains largely unknown. Methods: To explore the role of cytoplasmic Tet2 under CMS conditions, we established CMS mice model and detected the expression of RNA 5hmC by dot blot. We verified the interaction of Tet2 and its interacting protein by co-immunoprecipitation combined with mass spectrometry and screened downstream target genes by cluster analysis of Tet2 and upstream frameshift 1 (Upf1) interacting RNA. The expression of protein was detected by Western blot and the expression of the screened target genes was detected by qRT-PCR. Results: In this study, we found that increased cytoplasmic Tet2 expression under CMS conditions leads to increase in total RNA 5hmC modification. Tet2 interacted with the key non-sense-mediated mRNA decay (NMD) factor Upf1, regulated the stability of stress-related genes such as Unc5b mRNA, and might thereby affect neurodevelopment. Discussion: In summary, this study revealed that Tet2-mediated RNA 5hmC modification is involved in stress-related mRNA stability regulation and may serve as a potential therapeutic target for chronic stress-related diseases such as depression.

The mitochondrial Ahi1/GR participates the regulation on mtDNA copy numbers and brain ATP levels and modulates depressive behaviors in mice.

BACKGROUND: Previous studies have shown that depression is often accompanied by an increase in mtDNA copy number and a decrease in ATP levels; however, the exact regulatory mechanisms remain unclear. METHODS: In the present study, Western blot, cell knockdown, immunofluorescence, immunoprecipitation and ChIP-qPCR assays were used to detect changes in the Ahi1/GR-TFAM-mtDNA pathway in the brains of neuronal Abelson helper integration site-1 (Ahi1) KO mice and dexamethasone (Dex)-induced mice to elucidate the pathogenesis of depression. In addition, a rescue experiment was performed to determine the effects of regular exercise on the Ahi1/GR-TFAM-mtDNA-ATP pathway and depression-like behavior in Dex-induced mice and Ahi1 KO mice under stress. RESULTS: In this study, we found that ATP levels decreased and mitochondrial DNA (mtDNA) copy numbers increased in depression-related brain regions in Dex-induced depressive mice and Ahi1 knockout (KO) mice. In addition, Ahi1 and glucocorticoid receptor (GR), two important proteins related to stress and depressive behaviors, were significantly decreased in the mitochondria under stress. Intriguingly, GR can bind to the D-loop control region of mitochondria and regulate mitochondrial replication and transcription. Importantly, regular exercise significantly increased mitochondrial Ahi1/GR levels and ATP levels and thus improved depression-like behaviors in Dex-induced depressive mice but not in Ahi1 KO mice under stress. CONCLUSIONS: In summary, our findings demonstrated that the mitochondrial Ahi1/GR complex and TFAM coordinately regulate mtDNA copy numbers and brain ATP levels by binding to the D-loop region of mtDNA Regular exercise increases the levels of the mitochondrial Ahi1/GR complex and improves depressive behaviors. Video Abstract.

Phosphatidylserine, inflammation, and central nervous system diseases.

Phosphatidylserine (PS) is an anionic phospholipid in the eukaryotic membrane and is abundant in the brain. Accumulated studies have revealed that PS is involved in the multiple functions of the brain, such as activation of membrane signaling pathways, neuroinflammation, neurotransmission, and synaptic refinement. Those functions of PS are related to central nervous system (CNS) diseases. In this review, we discuss the metabolism of PS, the anti-inflammation function of PS in the brain; the alterations of PS in different CNS diseases, and the possibility of PS to serve as a therapeutic agent for diseases. Clinical studies have showed that PS has no side effects and is well tolerated. Therefore, PS and PS liposome could be a promising supplementation for these neurodegenerative and neurodevelopmental diseases.

Peripheral Interleukin-18 is negatively correlated with abnormal brain activity in patients with depression: a resting-state fMRI study.

BACKGROUND: Interleukin-18 (IL-18) may participate in the development of major depressive disorder, but the specific mechanism remains unclear. This study aimed to explore whether IL-18 correlates with areas of the brain associated with depression. METHODS: Using a case-control design, 68 subjects (34 patients and 34 healthy controls) underwent clinical assessment, blood sampling, and resting-state functional Magnetic Resonance Imaging (fMRI). The total Hamilton depression-17 (HAMD-17) score was used to assess depression severity. Enzyme-linked immunosorbent assay (ELISA) was used to detect IL-18 levels. Rest-state fMRI was conducted to explore spontaneous brain activity. RESULTS: The level of IL-18 was higher in patients with depression in comparison with healthy controls. IL-18 was negatively correlated with degree centrality of the left posterior cingulate gyrus in the depression patient group, but no correlation was found in the healthy control group. CONCLUSION: This study suggests the involvement of IL-18 in the pathophysiological mechanism for depression and interference with brain activity.

Ahi1 regulates serotonin production by the GR/ERß/TPH2 pathway involving sexual differences in depressive behaviors.

BACKGROUND: Depression is one of the most common psychiatric diseases. The monoamine transmitter theory suggests that neurotransmitters are involved in the mechanism of depression; however, the regulation on serotonin production is still unclear. We previously showed that Ahi1 knockout (KO) mice exhibited depression-like behavior accompanied by a significant decrease in brain serotonin. METHODS: In the present study, western blot, gene knockdown, immunofluorescence, dual-luciferase reporter assay, and rescue assay were used to detect changes in the Ahi1/GR/ERß/TPH2 pathway in the brains of male stressed mice and male Ahi1 KO mice to explain the pathogenesis of depression-like behaviors. In addition, E2 levels in the blood and brain of male and female mice were measured to investigate the effect on the ERß/TPH2 pathway and to reveal the mechanisms for the phenomenon of gender differences in depression-like behaviors. RESULTS: We found that the serotonin-producing pathway-the ERß/TPH2 pathway was inhibited in male stressed mice and male Ahi1 KO mice. We further demonstrated that glucocorticoid receptor (GR) as a transcription factor bound to the promoter of ERß that contains glucocorticoid response elements and inhibited the transcription of ERß. Our recent study had indicated that Ahi1 regulates the nuclear translocation of GR upon stress, thus proposing the Ahi1/GR/ERß/TPH2 pathway for serotonin production. Interestingly, female Ahi1 KO mice did not exhibit depressive behaviors, indicating sexual differences in depressive behaviors compared with male mice. Furthermore, we found that serum 17ß-estradiol (E2) level was not changed in male and female mice; however, brain E2 level significantly decreased in male but not female Ahi1 KO mice. Further, ERß agonist LY-500307 increased TPH2 expression and 5-HT production. Therefore, both Ahi1 and E2 regulate the ERß/TPH2 pathway and involve sexual differences in brain serotonin production and depressive behaviors. CONCLUSIONS: In conclusion, although it is unclear how Ahi1 controls E2 secretion in the brain, our findings demonstrate that Ahi1 regulates serotonin production by the GR/ERß/TPH2 pathway in the brain and possibly involves the regulation on sex differences in depressive behaviors. Video Abstract.

Tet Enzymes-Mediated DNA 5hmC Modification in Cerebral Ischemic and Hemorrhagic Injury.

5-Hydroxymethylcytosine (5hmC) has recently been found that plays an important role in many diseases; however, there are still few studies in the field of stroke. The purpose of this review is to introduce the influence and function of 5hmC in stroke, in order for more people can study it. In this review, we introduced the role of 5hmC in ischemia and hemorrhage stroke, and summarized the possible therapeutic prospects of 5hmC in stroke. In conclusion, we suggest that 5hmC may serve as a biomarker or therapeutic target for the treatment of stroke.

Interleukin-18 mediated inflammatory brain injury after intracerebral hemorrhage in male mice.

Interleukin-18 (IL-18), a pro-inflammatory cytokine, is thought to be associated with inflammation in many neurological diseases such as ischemic stroke and poststroke depression, but the role of IL-18 in inflammatory injury after intracerebral hemorrhage (ICH) remains unclear. In this study, we established the ICH model in male mice and found that IL-18 expression including protein and mRNA levels was significantly increased in brain tissues after ICH. Meanwhile, exogenous IL-18 exacerbated cerebral hematoma and neurological deficits following ICH. In the IL-18 knockout group, the size of hematoma and neurological functions after ICH was decreased compared with the wild-type group, suggesting the critical role of IL-18 on the modulation of brain injury after ICH. Importantly, exogenous IL-18 increased microglial activation in brain tissues after ICH. Furthermore, IL-18 knockout resulted in the reduction of activated microglia after ICH. These results indicated that IL-18 may regulate the inflammatory response after ICH through the activation of microglia. Thus, IL-18 is expected to be a promising therapeutic target for secondary brain injury after ICH.

ß2SP/TET2 complex regulates gene 5hmC modification after cerebral ischemia.

ßII spectrin (ß2SP) is encoded by Sptbn1 and is involved in the regulation of various cell functions. ß2SP contributes to the formation of the myelin sheath, which may be related to the mechanism of neuropathy caused by demyelination. As one of the main features of cerebral ischemia, demyelination plays a key role in the mechanism of cerebral ischemia injury. Here, we showed that ß2SP levels were increased, and this molecule interacted with TET2 after ischemic injury. Furthermore, we found that the level of TET2 was decreased in the nucleus when ß2SP was knocked out after oxygen and glucose deprivation (OGD), and the level of 5hmC was reduced in the OGD+ß2SP KO group. In contrast, the expression of ß2SP did not change in TET2 KO mice. In addition, the 5hmC sequencing results revealed that ß2SP can affect the level of 5hmC, the differentially hydroxymethylated region (DhMR) mainly related with the Calcium signalling pathway, cGMP-PKG signalling pathway, Wnt signalling pathway and Hippo signalling pathway. In summary, our results suggest that ß2SP could regulate the gene 5hmC by interacted with TET2 and will become a potential therapeutic target for ischemic stroke.

Stress modulates Ahi1-dependent nuclear localization of ten-eleven translocation protein 2.

Major depression disorder is one of the most common psychiatric diseases. Recent evidence supports that environmental stress affects gene expression and promotes the pathological process of depression through epigenetic mechanisms. Three ten-eleven translocation (Tet) enzymes are epigenetic regulators of gene expression that promote 5-hydroxymethylcytosine (5hmC) modification of genes. Here, we show that the loss of Tet2 can induce depression-like phenotypes in mice. Paradoxically, using the paradigms of chronic stress, such as chronic mild stress and chronic social defeat stress, we found that depressive behaviors were associated with increased Tet2 expression but decreased global 5hmC level in hippocampus. We examined the genome-wide 5hmC profile in the hippocampus of Tet2 knockout mice and identified 651 dynamically hydroxymethylated regions, some of which overlapped with known depression-associated loci. We further showed that chronic stress could induce the abnormal nuclear translocation of Tet2 protein from cytosol. Through Tet2 immunoprecipitation and mass spectrum analyses, we identified a cellular trafficking protein, Abelson helper integration site-1 (Ahi1), which could interact with Tet2 protein. Ahi1 knockout or knockdown caused the accumulation of Tet2 in cytosol. The reduction of Ahi1 protein under chronic stress explained the abnormal Ahi1-dependent nuclear translocation of Tet2. These findings together provide the evidence for a critical role of modulating Tet2 nuclear translocation in regulating stress response.

Triglyceride regulate ACE2 level through MTHFD1.

Obesity has been followed with interest as a risk factor for COVID-19, with triglycerides as one of four common criteria used to define obesity, which have been used to study the mechanism of obesity. In this study, we showed that angiotensin-converting enzyme-2 (ACE2) is widely expressed in the mouse body, including the kidney, spleen, brain, heart, lung, liver, and testis, and that ACE2 levels increased after a high-fat diet. The ACE2 levels were recorded at 0 days, 3 days, 7 days, and 14 days after a high-fat diet, and they increased at 14 days after high-fat diet initiation. In addition, triglyceride levels were also significantly increased at 14 days after high-fat diet initiation, but body weight was not changed. Furthermore, we examined the ACE2 levels in Calu3 cells (a lung cancer cell line) after triglyceride treatment, and the results indicated that ACE2 levels were increased at 25 µM and reached their peak at 200 µM. Finally, we found that the mRNA level of mthfd1 was significantly increased in the high-fat diet group. Given these findings, we hypothesize that triglycerides can regulate the expression of ACE2 and Mthfd1.

Inhibition of the CDK5/caspase-3 Pathway by p5-TAT Protects Hippocampal Neurogenesis and Alleviates Radiation-induced Cognitive Dysfunction.

Radiation-induced cognitive dysfunction is a common complication associated with cranial radiation therapy. Inhibition of hippocampal neurogenesis and proliferation plays a critical role in this complication. Relieving hippocampal apoptosis may significantly protect hippocampal neurogenesis and proliferation. Previous studies have demonstrated that hyperactivity of cyclin-dependent kinase 5 (Cdk5) is closely related to apoptosis. The exact molecular changes and function of Cdk5 in radiation-induced cognitive dysfunction are still not clear. Whether inhibition of Cdk5 and the relevant caspase-3 could improve hippocampal neurogenesis and ameliorate radiation-induced cognitive dysfunction needs further exploration. We hypothesized that inhibition of the Cdk5/caspase-3 pathway by p5-TAT could protect hippocampal neurogenesis and alleviate radiation-induced cognitive dysfunction. In our study, we reported that radiation induced hyperactivity of Cdk5 accompanied by elevation of the levels of cleaved caspase-3, a marker of neuronal apoptosis. Inhibition of hippocampal neurogenesis and proliferation as well as cognitive dysfunction was also observed. p5-TAT, a specific inhibitor of Cdk5, decreased the overactivation of Cdk5 without affecting the levels of Cdk5 activators. Additionally, this treatment suppressed the expression of cleaved caspase-3. We further demonstrated that p5-TAT treatment reduced hippocampal dysfunction and improved behavioral performance. Therefore, Cdk5 inhibition by the small peptide p5-TAT is a promising therapeutic strategy for radiation-induced cognitive dysfunction.

The Role of Tet2-mediated Hydroxymethylation in Poststroke Depression.

Poststroke depression (PSD) is a common complication of stroke and has long been a serious threat to human health. PSD greatly affects neurological recovery, quality of life and mortality. Recent studies have shown that 5-hydroxymethylcytosine (5hmC), an important epigenetic modification, is enriched in the brain and associated with many neurological diseases. However, its role in PSD is still unclear. In this study, middle cerebral artery occlusion (MCAO) and spatial restraint stress were used to successfully induce a PSD mouse model and resulted in reduced 5hmC levels, which were caused by Tet2. Furthermore, genome-wide analysis of 5hmC revealed that differentially hydroxymethylated regions (DhMRs) were associated with PSD. DhMRs were enriched among genes involved in the Wnt signaling pathway, neuron development and learning or memory. In particular,DhMRs were strongly enriched in genes with lymphoid enhancer factor 1 (LEF1) binding motifs. Finally, we demonstrated that decreases in TET2 expression in the brain caused PSD by decreasing Wnt/ß-catenin/LEF1 pathway signaling to promote inflammatory factor IL-18 expression. In conclusion, our data highlight the potential for 5hmC modification as a therapeutic target for PSD.

Ahi1 regulates the nuclear translocation of glucocorticoid receptor to modulate stress response.

Stress activates the nuclear translocation of glucocorticoid receptors (GR) to trigger gene expression. Abnormal GR levels can alter the stress responses in animals and therapeutic effects of antidepressants. Here, we reported that stress-mediated nuclear translocation of GR reduced Ahi1 in the stressed cells and mouse brains. Ahi1 interacts with GR to stabilize each other in the cytoplasm. Importantly, Ahi1 deficiency promotes the degradation of GR in the cytoplasm and reduced the nuclear translocation of GR in response to stress. Genetic depletion of Ahi1 in mice caused hyposensitivity to antidepressants under the stress condition. These findings suggest that AHI1 is an important regulator of GR level and may serve as a therapeutic target for stress-related disorders.

Epigenetic Mechanisms of Paternal Stress in Offspring Development and Diseases.

The major biological function of the sperm cell is to transmit the paternal genetic and epigenetic information to the embryo as well as the following offspring. Sperm has a unique epigenome. An increasing body of epidemiological study supports that paternal stress induced by environmental exposures and lifestyle can modulate the sperm epigenome (including histone modification, DNA methylation, and noncoding RNA expression), sperm-egg fusion, embryo development, and offspring health. Based on the existing literature, we have summarized the paternal exposure on sperm epigenome along with the representative phenotypes of offspring and the possible mechanism involved.

A Role of the Podoplanin-CLEC-2 Axis in Promoting Inflammatory Response After Ischemic Stroke in Mice.

C-type lectin-like receptor 2 (CLEC-2) is a platelet surface-activating receptor with the prominent involvement in platelet activation, which was found to be associated with the progression and prognosis of acute ischemic stroke patients. Although podoplanin is the only known endogenous ligand for CLEC-2, the role of podoplanin/CLEC-2 in cerebral ischemia injury was unclear. In this study, we examined their role by using a mouse middle cerebral artery occlusion (MCAO) model. The expression of CLEC-2 and podoplanin increased after ischemia/reperfusion (I/R) injury, peaked at 24 h, and then decreased gradually. Podoplanin and CLEC-2 co-localized mainly in the ischemia/reperfusion cortex and expressed on neurons and microglia. Anti-podoplanin antibody pretreatment reduced cerebral infarct volume from 52.67 ± 4.67 to 34.08 ± 6.04% (P < 0.05) and attenuated the neurological deficits during acute stage and recovery stage. Moreover, a significant decrease of IL-18 and IL-1ß was observed in the mice pretreated with the anti-podoplanin antibody. Our results demonstrate that the podoplanin-CLEC-2 axis might play an important role in cerebral ischemia/reperfusion injury in mice by promoting inflammatory reactions.

Prevalence of posttraumatic stress symptoms in health care workers after exposure to patients with COVID-19.

OBJECTIVE: To investigate the prevalence of posttraumatic stress symptoms (PTSS) of health care workers (HCWs) who were potentially or directly exposed to patients with coronavirus disease 2019 (COVID-19) in a non-core epidemic area of China. METHODS: Psychological conditions were evaluated by the multiple psychological evaluation scales in HCWs at the Affiliated Hospital of Xuzhou Medical University in Xuzhou City (a non-core epidemic area in China) during COVID-19 epidemic. According to the risk of exposure to COVID-19 patients, HCWs were divided into two groups: HCWs with high-risk (HHCW) group (who worked in wards for COVID-19 patients) and HCWs with low-risk (LHCW) group (who worked in wards for non-COVID-19 patients in the same hospital). The clinical data of psychological evaluation scales from HCWs were collected. RESULTS: A total of 171 HCWs were recruited in this study, with 94 (55.0%) HCWs in the HHCW group, and 77 (45.0%) HCWs in the LHCW group. Significant differences were observed in gender, work stress, job risk, and levels of fear and anxiety, and the depression between the two groups (P < 0.05). The incidence of PTSS was 28.7% in HHCW group, while the incidence of PTSS was 13.0% in LHCW group. The PTSS between the two groups was statistically significant (P < 0.05). Further logistic regression analysis displayed that the exposure to COVID-19, work stress and coping strategies were major risk factors associated with PTSS. CONCLUSIONS: This study demonstrated that HCWs in HHCW group had a higher chance of developing PTSS when compared with those in LHCW group. The HCWs who were exposed to COVID-19 patients had more stress and chronic stress-related disorders. Stress management should be provided to the first line HCWs who combat with COVID-19.

Re-defining the clinicopathological spectrum of neuronal intranuclear inclusion disease.

BACKGROUND: The rapidly increasing case reports revealed that neuronal intranuclear inclusion disease (NIID) had concomitant other system symptoms besides nervous system symptoms. In this study, we systematically evaluated the symptoms, signs, auxiliary examination, and pathological changes in different systems in NIID patients. METHODS: NIID patients were confirmed by examining GGC repeats in the NOTCH2NLC gene. Clinical data of NIID patients including symptoms, signs, and auxiliary examinations were collected for analysis. Ubiquitin and p62 were detected in different tissues from previous surgical samples. RESULTS: Fifty-one NIID patients from 17 families were included in this study. Except neurological symptoms, clinical manifestations from other systems were very notable and diverse. The proportions of different system symptoms were 88.2% in nervous system, 78.4% in respiratory system, 72.5% in circulatory system, 72.5% in locomotor system, 66.7% in urinary system, 64.7% in digestive system, 61.5% in reproductive system, and 50.0% in endocrine system. In addition, other common symptoms included sexual dysfunction (43.1%), pupil constriction (56.9%), blurred vision (51.0%), and hearing loss (23.5%). Ubiquitin and p62-positive cells were found in different tissues and systems in 24 NIID patients with previous surgery. Initial symptoms of NIID and median onset age in different systems also revealed system heterogeneity of NIID. INTERPRETATION: For the first time, we systematically demonstrated that NIID is a heterogeneous and systemic neurodegenerative disease by providing clinical and pathological evidence. In addition to the nervous system, the clinical symptomatic and pathological spectrum of NIID has been extended to almost all systems.