c-Jun N-terminal kinase (JNK) is a member of the mitogen-activated protein kinase family and controls various physiological processes including apoptosis. A specific upstream activator of JNKs is the mitogen-activated protein kinase kinase 7 (MKK7). It has been reported that MKK7-JNK signaling plays an important regulatory role in neural development, however, post-developmental functions in the nervous system have not been elucidated. In this study, we generated neuron-specific Mkk7 knockout mice (MKK7 cKO), which impaired constitutive activation of JNK in the nervous system. MKK7 cKO mice displayed impaired circadian behavioral rhythms and decreased locomotor activity. MKK7 cKO mice at 8 months showed motor dysfunctions such as weakness of hind-limb and gait abnormality in an age-dependent manner. Axonal degeneration in the spinal cord and muscle atrophy were also observed, along with accumulation of the axonal transport proteins JNK-interacting protein 1 and amyloid beta precursor protein in the brains and spinal cords of MKK7 cKO mice. Thus, the MKK7-JNK signaling pathway plays important roles in regulating circadian rhythms and neuronal maintenance in the adult nervous system.

Glutamate released by activated microglia induces excitotoxic neuronal death, which likely contributes to non-cell autonomous neuronal death in neurodegenerative diseases, including amyotrophic lateral sclerosis and Alzheimer's disease. Although both blockade of glutamate receptors and inhibition of microglial activation are the therapeutic candidates for these neurodegenerative diseases, glutamate receptor blockers also perturbed physiological and essential glutamate signals, and inhibitors of microglial activation suppressed both neurotoxic/neuroprotective roles of microglia and hardly affected disease progression. We previously demonstrated that activated microglia release a large amount of glutamate specifically through gap junction hemichannel. Hence, blockade of gap junction hemichannel may be potentially beneficial in treatment of neurodegenerative diseases.

Two motor neuron diseases, amyotrophic lateral sclerosis (ALS) and spinal muscular atrophy (SMA), are caused by distinct genes involved in RNA metabolism, TDP-43 and FUS/TLS, and SMN, respectively. However, whether there is a shared defective mechanism in RNA metabolism common to these two diseases remains unclear. Here, we show that TDP-43 and FUS/TLS localize in nuclear Gems through an association with SMN, and that all three proteins function in spliceosome maintenance. We also show that in ALS, Gems are lost, U snRNA levels are up-regulated and spliceosomal U snRNPs abnormally and extensively accumulate in motor neuron nuclei, but not in the temporal lobe of FTLD with TDP-43 pathology. This aberrant accumulation of U snRNAs in ALS motor neurons is in direct contrast to SMA motor neurons, which show reduced amounts of U snRNAs, while both have defects in the spliceosome. These findings indicate that a profound loss of spliceosome integrity is a critical mechanism common to neurodegeneration in ALS and SMA, and may explain cell-type specific vulnerability of motor neurons.

Microglia are the principal phagocytes that clear cell debris in the central nervous system (CNS). This raises the question, which cells remove cell debris when microglial phagocytic activity is impaired. We addressed this question using Siglechdtr mice, which enable highly specific ablation of microglia. Non-microglial mononuclear phagocytes, such as CNS-associated macrophages and circulating inflammatory monocytes, did not clear microglial debris. Instead, astrocytes were activated, exhibited a pro-inflammatory gene expression profile, and extended their processes to engulf microglial debris. This astrocytic phagocytosis was also observed in Irf8-deficient mice, in which microglia were present but dysfunctional. RNA-seq demonstrated that even in a healthy CNS, astrocytes express TAM phagocytic receptors, which were the main astrocytic phagocytic receptors for cell debris in the above experiments, indicating that astrocytes stand by in case of microglial impairment. This compensatory mechanism may be important for the maintenance or prolongation of a healthy CNS.

Amyotrophic lateral sclerosis (ALS), also known as motor neuron disease as well as Lou Gehrig's disease, is a progressive neurological disorder selectively affecting motor neurons with no currently known cure. Around 20% of the familial ALS cases arise from dominant mutations in the sod1 gene encoding superoxide dismutase1 (SOD1) enzyme. Aggregation of mutant SOD1 in familial cases and of wild-type SOD1 in at least some sporadic ALS cases is one of the known causes of the disease. Riluzole, approved in 1995 and edaravone in 2017 remain the only drugs with limited therapeutic benefits.

Previously, we showed that cryo fixation of adult mouse brain tissue gave a truer representation of brain ultrastructure in comparison with a standard chemical fixation method (Korogod et al., 2015). Extracellular space matched physiological measurements, there were larger numbers of docked vesicles and less glial coverage of synapses and blood capillaries. Here, using the same preservation approaches, we compared the morphology of dendritic spines. We show that the length of the spine and the volume of its head is unchanged; however, the spine neck width is thinner by more than 30% after cryo fixation. In addition, the weak correlation between spine neck width and head volume seen after chemical fixation was not present in cryo-fixed spines. Our data suggest that spine neck geometry is independent of the spine head volume, with cryo fixation showing enhanced spine head compartmentalization and a higher predicted electrical resistance between spine head and parent dendrite.

The cytoplasmic aggregation of TAR DNA binding protein-43 (TDP-43), also known as TDP-43 pathology, is the pathological hallmark of amyotrophic lateral sclerosis (ALS). However, the mechanism underlying TDP-43 cytoplasmic mislocalization and subsequent aggregation remains unclear. Here, we show that TDP-43 dimerization/multimerization is impaired in the postmortem brains and spinal cords of patients with sporadic ALS and that N-terminal dimerization-deficient TDP-43 consists of pathological inclusion bodies in ALS motor neurons. Expression of N-terminal dimerization-deficient mutant TDP-43 in Neuro2a cells and induced pluripotent stem cell-derived motor neurons recapitulates TDP-43 pathology, such as Nxf1-dependent cytoplasmic mislocalization and aggregate formation, which induces seeding effects. Furthermore, TDP-DiLuc, a bimolecular luminescence complementation reporter assay, could detect decreased N-terminal dimerization of TDP-43 before TDP-43 pathological changes caused by the transcription inhibition linked to aberrant RNA metabolism in ALS. These findings identified TDP-43 monomerization as a critical determinant inducing TDP-43 pathology in ALS.

Neuroinflammation substantially contributes to the pathology of Alzheimer's disease (AD), the most common form of dementia. Studies have reported that nuclear factor erythroid 2-related factor 2 (Nrf2) attenuates neuroinflammation in the mouse models of neurodegenerative diseases, however, the detailed mechanism remains unclear.

Successful recovery from neuronal damage requires a huge energy supply, which is provided by mitochondria. However, the physiological relevance of mitochondrial dynamics in damaged neurons in vivo is poorly understood. To address this issue, we established unique bacterial artificial chromosome transgenic (BAC Tg) mice, which develop and function normally, but in which neuronal injury induces labelling of mitochondria with green fluorescent protein (GFP) and expression of cre recombinase. GFP-labelled mitochondria in BAC Tg mice appear shorter in regenerating motor axons soon after nerve injury compared with mitochondria in non-injured axons, suggesting the importance of increased mitochondrial fission during the early phase of nerve regeneration. Crossing the BAC Tg mice with mice carrying a floxed dynamin-related protein 1 gene (Drp1), which is necessary for mitochondrial fission, ablates mitochondrial fission specifically in injured neurons. Injury-induced Drp1-deficient motor neurons show elongated or abnormally gigantic mitochondria, which have impaired membrane potential and axonal transport velocity during the early phase after injury, and eventually promote neuronal death. Our in vivo data suggest that acute and prominent mitochondrial fission during the early stage after nerve injury is an adaptive response and is involved in the maintenance of mitochondrial and neuronal integrity to prevent neurodegeneration.

Dominant mutations in superoxide dismutase 1 (SOD1) cause degeneration of motor neurons in a subset of inherited amyotrophic lateral sclerosis (ALS). The pathogenetic process mediated by misfolded and/or aggregated mutant SOD1 polypeptides is hypothesized to be suppressed by protein refolding. This genetic study is aimed to test whether mutant SOD1-mediated ALS pathology recapitulated in mice could be alleviated by overexpressing a longevity-related deacetylase SIRT1 whose substrates include a transcription factor heat shock factor 1 (HSF1), the master regulator of the chaperone system.

Dominant mutations in Cu/Zn-superoxide dismutase (SOD1) gene cause a familial form of amyotrophic lateral sclerosis (SOD1-ALS) with accumulation of misfolded SOD1 proteins as intracellular inclusions in spinal motor neurons. Oligomerization of SOD1 via abnormal disulfide crosslinks has been proposed as one of the misfolding pathways occurring in mutant SOD1; however, the pathological relevance of such oligomerization in the SOD1-ALS cases still remains obscure.

Transcription-coupled nucleotide excision repair (TC-NER) is initiated by the stalling of elongating RNA polymerase II (RNAPIIo) at DNA lesions. The ubiquitination of RNAPIIo in response to DNA damage is an evolutionarily conserved event, but its function in mammals is unknown. Here, we identified a single DNA damage-induced ubiquitination site in RNAPII at RPB1-K1268, which regulates transcription recovery and DNA damage resistance. Mechanistically, RPB1-K1268 ubiquitination stimulates the association of the core-TFIIH complex with stalled RNAPIIo through a transfer mechanism that also involves UVSSA-K414 ubiquitination. We developed a strand-specific ChIP-seq method, which revealed RPB1-K1268 ubiquitination is important for repair and the resolution of transcriptional bottlenecks at DNA lesions. Finally, RPB1-K1268R knockin mice displayed a short life-span, premature aging, and neurodegeneration. Our results reveal RNAPII ubiquitination provides a two-tier protection mechanism by activating TC-NER and, in parallel, the processing of DNA damage-stalled RNAPIIo, which together prevent prolonged transcription arrest and protect against neurodegeneration.

Adult neurogenesis rarely occurs in the enteric nervous system (ENS). In this study, we demonstrated that, after intestinal myenteric plexus (MP) ablation with benzalkonium chloride (BAC), adult neurogenesis in the ENS was significantly induced in c-kit loss-of-function mutant mice (W/W(v)). Almost all neurons and fibers in the MP disappeared after BAC treatment. However, 1 week after ablation, substantial penetration of nerve fibers from the non-damaged area was observed in the MP, longitudinal muscle and subserosal layers in both wildtype and W/W(v) mice. Two weeks after BAC treatment, in addition to the penetrating fibers, a substantial number of ectopic neurons appeared in the subserosal and longitudinal muscle layers of W/W(v) mice, whereas only a few ectopic neurons appeared in wildtype mice. Such ectopic neurons expressed either excitatory or inhibitory intrinsic motor neuron markers and formed ganglion-like structures, including glial cells, synaptic vesicles and basal lamina. Furthermore, oral administration of imatinib, an inhibitor of c-Kit and an anticancer agent for gastrointestinal stromal tumors, markedly induced appearance of ectopic neurons after BAC treatment, even in wildtype mice. These results suggest that adult neurogenesis in the ENS is negatively regulated by c-Kit signaling in vivo.

The dura mater contains abundant macrophages whose functions remain largely elusive. Recent studies have demonstrated the origin, as well as the gene expression pattern, of dural macrophages (dMΦs). However, their histological features have not been explored yet. In this study, we performed immunohistochemistry and electron microscopy to elucidate their precise morphology, localization, and postnatal development in mice. We found that the morphology, as well as the localization, of dMΦs changed during postnatal development. In neonatal mice, dMΦ exhibited an amoeboid morphology. During postnatal development, their cell bodies elongated longitudinally and became aligned along dural blood vessels. In adulthood, nearly half of the dMΦs aligned along blood vessel networks. However, most of these cells were not directly attached to vessels; pericytes and fibroblasts interposed between dMΦs and vessels. This morphological information may provide further indications for the functional significance of dMΦs.

Microglia-mediated neuroinflammation has been implicated in the pathogenesis of Alzheimer's disease (AD). Although microglia in aging and neurodegenerative disease model mice show a loss of homeostatic phenotype and activation of disease-associated microglia (DAM), a correlation between those phenotypes and the degree of neuronal cell loss has not been clarified. In this study, we performed RNA sequencing of microglia isolated from three representative neurodegenerative mouse models, AppNL-G-F/NL-G-F with amyloid pathology, rTg4510 with tauopathy, and SOD1G93A with motor neuron disease by magnetic activated cell sorting. In parallel, gene expression patterns of the human precuneus with early Alzheimer's change (n = 11) and control brain (n = 14) were also analyzed by RNA sequencing. We found that a substantial reduction of homeostatic microglial genes in rTg4510 and SOD1G93A microglia, whereas DAM genes were uniformly upregulated in all mouse models. The reduction of homeostatic microglial genes was correlated with the degree of neuronal cell loss. In human precuneus with early AD pathology, reduced expression of genes related to microglia- and oligodendrocyte-specific markers was observed, although the expression of DAM genes was not upregulated. Our results implicate a loss of homeostatic microglial function in the progression of AD and other neurodegenerative diseases. Moreover, analyses of human precuneus also suggest loss of microglia and oligodendrocyte functions induced by early amyloid pathology in human.

Cytoplasmic inclusion of TAR DNA-binding protein 43 (TDP-43) is a pathological hallmark of amyotrophic lateral sclerosis (ALS) and a subtype of frontotemporal lobar degeneration (FTLD). Recent studies have suggested that the formation of cytoplasmic TDP-43 aggregates is dependent on a liquid-liquid phase separation (LLPS) mechanism. However, it is unclear whether TDP-43 pathology is induced through a single intracellular mechanism such as LLPS. To identify intracellular mechanisms responsible for TDP-43 aggregation, we established a TDP-43 aggregation screening system using a cultured neuronal cell line stably expressing EGFP-fused TDP-43 and a mammalian expression library of the inherited ALS/FTLD causative genes, and performed a screening. We found that microtubule-related proteins (MRPs) and RNA-binding proteins (RBPs) co-aggregated with TDP-43. MRPs and RBPs sequestered TDP-43 into the cytoplasmic aggregates through distinct mechanisms, such as microtubules and LLPS, respectively. The MRPs-induced TDP-43 aggregates were co-localized with aggresomal markers and dependent on histone deacetylase 6 (HDAC6), suggesting that aggresome formation induced the co-aggregation. However, the MRPs-induced aggregates were not affected by 1,6-hexanediol, an LLPS inhibitor. On the other hand, the RBPs-induced TDP-43 aggregates were sensitive to 1,6-hexanediol, but not dependent on microtubules or HDAC6. In sporadic ALS patients, approximately half of skein-like TDP-43 inclusions were co-localized with HDAC6, but round and granular type inclusion were not. Moreover, HDAC6-positive and HDAC6-negative inclusions were found in the same ALS patient, suggesting that the two distinct pathways are both involved in TDP-43 pathology. Our findings suggest that at least two distinct pathways (i.e., aggresome formation and LLPS) are involved in inducing the TDP-43 pathologies.

Intracellular mislocalization of TAR DNA-binding protein 43 (TDP-43), a nuclear DNA/RNA-binding protein involved in RNA metabolism, is a pathological hallmark of amyotrophic lateral sclerosis (ALS). Although the aggregation-prone, TDP-43 C-terminal domain is widely considered as a key component of TDP-43 pathology in ALS, recent studies including ours suggest that TDP-43 N-terminal fragments (TDP-∆C) may also contribute to the motor dysfunction in ALS. However, the specific pathological functions of TDP-43 N-terminal fragments in mice have not been elucidated. Here, we established TDP-∆C knock-in mice missing a part of exon 6 of murine Tardbp gene, which encodes the C-terminal region of TDP-43. Homozygous TDP-∆C mice showed embryonic lethality, indicating that the N-terminal domain of TDP-43 alone is not sufficient for normal development. In contrast, heterozygous TDP-∆C mice developed normally but exhibited age-dependent mild motor dysfunction with a loss of C-boutons, large cholinergic synaptic terminals on spinal α-motor neurons. TDP-∆C protein broadly perturbed gene expression in the spinal cords of aged heterozygous TDP-∆C mice, including downregulation of Notch1 mRNA. Moreover, the level of Notch1 mRNA was suppressed both by TDP-43 depletion and TDP-∆C expression in Neuro2a cells. Decreased Notch1 mRNA expression in aged TDP-∆C mice was associated with the age-dependent motor dysfunction and loss of Akt surviving signal. Our findings indicate that the N-terminal region of TDP-43 derived from TDP-∆C induces the age-dependent motor dysfunction associated with impaired Notch1-Akt axis in mice.

The organelle contact site of the endoplasmic reticulum and mitochondria, known as the mitochondria-associated membrane (MAM), is a multifunctional microdomain in cellular homeostasis. We previously reported that MAM disruption is a common pathological feature in amyotrophic lateral sclerosis (ALS); however, the precise role of MAM in ALS was uncovered. Here, we show that the MAM is essential for TANK-binding kinase 1 (TBK1) activation under proteostatic stress conditions. A MAM-specific E3 ubiquitin ligase, autocrine motility factor receptor, ubiquitinated nascent proteins to activate TBK1 at the MAM, which results in ribosomal protein degradation. MAM or TBK1 deficiency under proteostatic stress conditions resulted in increased cellular vulnerability in vitro and motor impairment in vivo. Thus, MAM disruption exacerbates proteostatic stress via TBK1 inactivation in ALS. Our study has revealed a proteostatic mechanism mediated by the MAM-TBK1 axis, highlighting the physiological importance of the organelle contact sites.

Mitochondria undergo morphological changes through fusion and fission for their quality control, which are vital for neuronal function. In this study, we examined three-dimensional morphologies of mitochondria in motor neurons under normal, nerve injured, and nerve injured plus fission-impaired conditions using the focused ion beam/scanning electron microscopy (FIB/SEM), because the FIB/SEM technology is a powerful tool to demonstrate both 3D images of whole organelle and the intra-organellar structure simultaneously. Crossing of dynamin-related protein 1 (Drp1) gene-floxed mice with neuronal injury-specific Cre driver mice, Atf3:BAC Tg mice, allowed for Drp1 ablation specifically in injured neurons. FIB/SEM analysis demonstrated that somatic mitochondrial morphologies in motor neurons were not altered before or after nerve injury. However, the fission impairment resulted in prominent somatic mitochondrial enlargement, which initially induced complex morphologies with round regions and long tubular processes, subsequently causing a decrease in the number of processes and further enlargement of the round regions, which eventually resulted in big spheroidal mitochondria without processes. The abnormal mitochondria exhibited several degradative morphologies: local or total cristae collapse, vacuolization, and mitophagy. These suggest that mitochondrial fission is crucial for maintaining mitochondrial integrity in injured motor neurons, and multiple forms of mitochondria degradation may accelerate neuronal degradation.

Differential vulnerability among motor neuron (MN) subtypes is a fundamental feature of amyotrophic lateral sclerosis (ALS): fast-fatigable (FF) MNs are more vulnerable than fast fatigue-resistant (FR) or slow (S) MNs. The reason for this selective vulnerability remains enigmatic. We report here that the extracellular matrix (ECM) protein osteopontin (OPN) is selectively expressed by FR and S MNs and ALS-resistant motor pools, whereas matrix metalloproteinase-9 (MMP-9) is selectively expressed by FF MNs. OPN is secreted and accumulated as extracellular granules in ECM in three ALS mouse models and a human ALS patient. In SOD1(G93A) mice, OPN/MMP-9 double positivity marks remodeled FR and S MNs destined to compensate for lost FF MNs before ultimately dying. Genetic ablation of OPN in SOD1(G93A) mice delayed disease onset but then accelerated disease progression. OPN induced MMP-9 up-regulation via αvβ3 integrin in ChAT-expressing Neuro2a cells, and also induced CD44-mediated astrocyte migration and microglial phagocytosis in a non-cell-autonomous manner. Our results demonstrate that OPN expressed by FR/S MNs is involved in the second-wave neurodegeneration by up-regulating MMP-9 through αvβ3 integrin in the mouse model of ALS. The differences in OPN/MMP-9 expression profiles in MN subsets partially explain the selective MN vulnerability in ALS.