Dorsomedial prefrontal hypoexcitability underlies lost empathy in frontotemporal dementia.

Empathic function is essential for the well-being of social species. Empathy loss is associated with various brain disorders and represents arguably the most distressing feature of frontotemporal dementia (FTD), a leading form of presenile dementia. The neural mechanisms are unknown. We established an FTD mouse model deficient in empathy and observed that aged somatic transgenic mice expressing GGGGCC repeat expansions in C9orf72, a common genetic cause of FTD, exhibited blunted affect sharing and failed to console distressed conspecifics by affiliative contact. Distress-induced consoling behavior activated the dorsomedial prefrontal cortex (dmPFC), which developed profound pyramidal neuron hypoexcitability in aged mutant mice. Optogenetic dmPFC inhibition attenuated affect sharing and other-directed consolation in wild-type mice, whereas chemogenetically enhancing dmPFC excitability rescued empathy deficits in mutant mice, even at advanced ages when substantial cortical atrophy had occurred. These results establish cortical hypoexcitability as a pathophysiological basis of empathy loss in FTD and suggest a therapeutic strategy.

Prevention of mitochondrial impairment by inhibition of protein phosphatase 1 activity in amyotrophic lateral sclerosis.

Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease caused by progressive loss of motor neurons (MNs) and subsequent muscle weakness. These pathological features are associated with numerous cellular changes, including alteration in mitochondrial morphology and function. However, the molecular mechanisms associating mitochondrial structure with ALS pathology are poorly understood. In this study, we found that Dynamin-related protein 1 (Drp1) was dephosphorylated in several ALS models, including those with SOD1 and TDP-43 mutations, and the dephosphorylation was mediated by the pathological induction of protein phosphatase 1 (PP1) activity in these models. Suppression of the PP1-Drp1 cascade effectively prevented ALS-related symptoms, including mitochondrial fragmentation, mitochondrial complex I impairment, axonal degeneration, and cell death, in primary neuronal culture models, iPSC-derived human MNs, and zebrafish models in vivo. These results suggest that modulation of PP1-Drp1 activity may be a therapeutic target for multiple pathological features of ALS.

C9ORF72-ALS/FTD-associated poly(GR) binds Atp5a1 and compromises mitochondrial function in vivo.

The GGGGCC repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). However, it is not known which dysregulated molecular pathways are primarily responsible for disease initiation or progression. We established an inducible mouse model of poly(GR) toxicity in which (GR)80 gradually accumulates in cortical excitatory neurons. Low-level poly(GR) expression induced FTD/ALS-associated synaptic dysfunction and behavioral abnormalities, as well as age-dependent neuronal cell loss, microgliosis and DNA damage, probably caused in part by early defects in mitochondrial function. Poly(GR) bound preferentially to the mitochondrial complex V component ATP5A1 and enhanced its ubiquitination and degradation, consistent with reduced ATP5A1 protein level in both (GR)80 mouse neurons and patient brains. Moreover, inducing ectopic Atp5a1 expression in poly(GR)-expressing neurons or reducing poly(GR) level in adult mice after disease onset rescued poly(GR)-induced neurotoxicity. Thus, poly(GR)-induced mitochondrial defects are a major driver of disease initiation in C9ORF72-related ALS/FTD.

Partial inhibition of the overactivated Ku80-dependent DNA repair pathway rescues neurodegeneration in C9ORF72-ALS/FTD.

GGGGCC (G4C2) repeat expansion in C9ORF72 is the most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). One class of major pathogenic molecules in C9ORF72-ALS/FTD is dipeptide repeat proteins such as poly(GR), whose toxicity has been well documented in cellular and animal models. However, it is not known how poly(GR) toxicity can be alleviated, especially in patient neurons. Using Drosophila as a model system in an unbiased genetic screen, we identified a number of genetic modifiers of poly(GR) toxicity. Surprisingly, partial loss of function of Ku80, an essential DNA repair protein, suppressed poly(GR)-induced retinal degeneration in flies. Ku80 expression was greatly elevated in flies expressing poly(GR) and in C9ORF72 iPSC-derived patient neurons. As a result, the levels of phosphorylated ATM and P53 as well as other downstream proapoptotic proteins such as PUMA, Bax, and cleaved caspase-3 were all significantly increased in C9ORF72 patient neurons. The increase in the levels of Ku80 and some downstream signaling proteins was prevented by CRISPR-Cas9-mediated deletion of expanded G4C2 repeats. More importantly, partial loss of function of Ku80 in these neurons through CRISPR/Cas9-mediated ablation or small RNAs-mediated knockdown suppressed the apoptotic pathway. Thus, partial inhibition of the overactivated Ku80-dependent DNA repair pathway is a promising therapeutic approach in C9ORF72-ALS/FTD.

Physiological and pathological significance of dynamin-related protein 1 (drp1)-dependent mitochondrial fission in the nervous system.

Mitochondria are essential for proper neuronal morphogenesis and functions, as they are the major source of energy for neural development. The dynamic morphology of mitochondria determines the key functions of mitochondria. Several regulatory proteins such as dynamin-related protein 1 (Drp1) are required to maintain mitochondrial morphology via a balance between continuous fusion and fission. Activity of Drp1, a key regulator in mitochondrial fission, is modulated by multiple post-translation modifications and receptor interactions. In addition, numerous researches have revealed that the regulation of Drp1 activity and mitochondrial dynamics is closely associated with several neurodegenerative diseases such as Alzheimer's and Parkinson's diseases. In this article, we concisely review the recent findings about the biological importance of Drp1-mediated mitochondrial fission in neurons under physiological and pathological conditions.

Caffeine enhances osteoclast differentiation and maturation through p38 MAP kinase/Mitf and DC-STAMP/CtsK and TRAP pathway.

The consumption of caffeine from some common beverages has been associated with low bone mass by inducing urinary calcium loss and deceasing bone mineral density. However, the effect of caffeine on osteoclast differentiation is still unclear. Here, we demonstrate that caffeine directly enhances osteoclast differentiation and maturation. TRAP staining showed that the number of larger (>100 µm) osteoclastic cells as well as of TRAP-positive multinucleated cells was increased by caffeine treatment. Among the MAP kinases, caffeine specifically activated p38 MAP kinase, which in turn, controlled osteoclast differentiation and maturation. This is evidenced by the abolishment of activated p38 MAP kinase by pretreatment with SB203580, a p38-specific inhibitor, resulting in suppressed osteoclast differentiation and maturation that should be increased by caffeine. Caffeine significantly induced the expression of Mitf and pretreatment with SB203580 markedly suppressed the expression of Mitf induced by caffeine. Whereas it failed to regulate the expression of NFATc1 and Oscar, the expressions of Cathepsin K and TRAP were induced by caffeine treatment in primary preosteoclasts. Real-time PCR and luciferase assays showed that the increase of osteoclastic cell-cell fusion by caffeine was through the transcriptional up-regulation of DC-STAMP expression but not of Atp6v0d2. These results strongly suggest that caffeine directly enhances osteoclast differentiation and maturation through p38 MAP kinase activation, thus inducing Mitf expression and transcriptional activation of DC-STAMP, and finally CtsK and TRAP.

Expression of ezrin in subventricular zone neural stem cells and their progeny in adult and developing mice.

Ezrin is a member of the ezrin-radixin-moesin (ERM) family of proteins, which link the cytoskeleton and cell membrane. ERM proteins are involved in pivotal cellular functions including cell-matrix recognition, cell-cell communication, and cell motility. Several recent studies have shown that ERM proteins are expressed in specific cell types of the adult rostral migratory stream (RMS). In this study, we found that ERM proteins are expressed highly in the early postnatal RMS and the ventricular zone of embryonic cerebral cortex, suggesting that these proteins may be expressed by neural progenitors. Furthermore, whereas ezrin previously was found to be expressed exclusively by astrocytes of the adult RMS, we found that ezrin-expressing cells also expressed the markers for indicating neuroblasts in vivo and in vitro, and that ezrin expression by neuroblasts decreases progressively as neuroblasts migrate. Using in vitro differentiation of adult neural stem cells, we found that ezrin is expressed by neural stem cells and their progeny (neuroblasts and astrocytes), but not by oligodendrocytic progeny. Collectively our findings demonstrate that adult neural stem cells and neuroblasts express ezrin and that ezrin may be involved in intracellular actin remodeling.

Drp1-mediated mitochondrial dynamics and survival of developing chick motoneurons during the period of normal programmed cell death.

Mitochondrial morphology is dynamically remodeled by fusion and fission in neurons, and this process is implicated in nervous system development and pathology. However, the mechanism by which mitochondrial dynamics influence neuronal development is less clear. In this study, we found that the length of mitochondria is progressively reduced during normal development of chick embryo motoneurons (MNs), a process partly controlled by a fission-promoting protein, dynamin-related protein 1 (Drp1). Suppression of Drp1 activity by gene electroporation of dominant-negative mutant Drp1 in a subset of developing MNs increased mitochondrial length in vivo, and a greater proportion of Drp1-suppressed MNs underwent programmed cell death (PCD). By contrast, the survival of nontransfected MNs in proximity to the transfected MNs was significantly increased, suggesting that the suppression of Drp1 confers disadvantage during the competition for limited survival signals. Because we also monitored perturbation of neurite outgrowth and mitochondrial membrane depolarization following Drp1 suppression, we suggest that impairments of ATP production and axonal growth may be downstream factors that influence the competition of MNs for survival. Collectively, these results indicate that mitochondrial dynamics are required for normal axonal development and competition-dependent MN PCD.

Differential expression of BNIP family members of BH3-only proteins during the development and after axotomy in the rat.

The BNIPs (BCL2 and adenovirus E1B 19 kDa interacting proteins) are a subfamily of BCL2 family proteins typically containing a single BCL2 homology 3 (BH3) domain. BNIPs exert important roles in two major degradation processes in cells - apoptosis and autophagy. Although it is known that the function of BNIPs is transcriptionally regulated under hypoxic conditions in tumors, their regulation in the developing brain and neurons following the induction of apoptosis/autophagy is largely unknown. In this study, we demonstrate that three members of the BNIP family, BNIP1, BNIP3 and BNIP3L, are expressed in the developing brain with distinct brain region specificity. BNIP3 mRNA was especially enriched in the entorhinal cortex, raising a possibility that it may have additional biological functions in addition to its apoptotic and autophagic functions. Following starvation-induced autophagy induction, BNIP1 mRNA was selectively increased in cultured neurons. However, the apoptogenic chemical staurosporine failed to modulate the expression of BNIPs, which is in contrast to the marked induction of all BNIPs by glucose-oxygen deprivation. Finally, neonatal nerve axotomy, which triggers apoptosis in motoneurons, selectively enhanced BNIP3 mRNA expression. Collectively, these results suggest that the expression of BNIPs is differentially regulated depending on the stimuli, and BNIPs may exert unique biological functions.

PI3Kγ is required for NMDA receptor-dependent long-term depression and behavioral flexibility.

Phosphatidylinositol 3-kinase (PI3K) has been implicated in synaptic plasticity and other neural functions in the brain. However, the role of individual PI3K isoforms in the brain is unclear. We investigated the role of PI3Kγ in hippocampal-dependent synaptic plasticity and cognitive functions. We found that PI3Kγ has a crucial and specific role in NMDA receptor (NMDAR)-mediated synaptic plasticity at mouse Schaffer collateral-commissural synapses. Both genetic deletion and pharmacological inhibition of PI3Kγ disrupted NMDAR long-term depression (LTD) while leaving other forms of synaptic plasticity intact. Accompanying this physiological deficit, the impairment of NMDAR LTD by PI3Kγ blockade was specifically correlated with deficits in behavioral flexibility. These findings suggest that a specific PI3K isoform, PI3Kγ, is critical for NMDAR LTD and some forms of cognitive function. Thus, individual isoforms of PI3Ks may have distinct roles in different types of synaptic plasticity and may therefore influence various kinds of behavior.

Induction of ezrin-radixin-moesin molecules after cryogenic traumatic brain injury of the mouse cortex.

Traumatic brain injury promotes rapid induction of microglial cells and infiltration of peripheral macrophages to the injury sites. Such inflammatory responses are mediated by the activation and migration of immune cells, which are influenced by the actin cytoskeleton remodeling. In this study, we observed that the phosphorylation and expressions of ezrin-radixin-moesin (ERM) proteins, which are linkers for cell surface with actin cytoskeleton, are induced in the activated microglia/macrophages, whereas ERM molecules are only marginally expressed in quiescent microglia in the normal brain. These results suggest that ERM activation in the injury penumbra is implicated in the inflammatory immune responses after traumatic brain injury.

Nuclear translocation of anamorsin during drug-induced dopaminergic neurodegeneration in culture and in rat brain.

Anamorsin, also called cytokine-induced apoptosis inhibitor 1 (CIAPIN1), was recently identified to confer resistance to apoptosis induced by growth factor deprivation and to be indispensible for hematopoiesis. Recently, it was demonstrated that anamorsin is also widely distributed in both fetal and adult tissues. In this study, we evaluated the tissue distribution of anamorsin in the central nervous system (CNS) during development. In situ hybridization and immunoblot analyses revealed that anamorsin mRNA and protein were both highly and widely expressed in various regions of the CNS, including the cerebral cortex, hippocampus, midbrain, cerebellum, medulla, and spinal cord. Based on these findings, we examined its cellular localization during drug-induced neurodegeneration in MN9D dopaminergic cells. Both immunocytochemical localization and immunoblot analyses indicated that cytosolic anamorsin was translocated into the nucleus in a time-dependent manner following treatment with a reactive oxygen species (ROS)-inducing drug, 6-hydroxydopamine (6-OHDA). Treatment of cells with the apoptosis-inducing reagent, staurosporine, did not appear to cause translocation of anamorsin into the nucleus. When cells were treated with the nuclear export inhibitor, Leptomycin B, alone or with 6-OHDA, nuclear anamorsin levels increased, indicating that nuclear influx and efflux of anamorsin are regulated by 6-OHDA treatment. In rat brain injected with 6-OHDA, nuclear translocation of anamorsin was identified in certain tyrosine hydroxylase (TH)-positive neurons as well as TH-negative cells. Furthermore, treatment of MN9D cells with hydrogen peroxide or ROS-inducing trace metals caused nuclear translocation of anamorsin. Taken together, our data indicate that nuclear translocation of anamorsin is a ROS-dependent event and may participate in the regulation of transcription of critical molecules during dopaminergic neurodegeneration.

Effect of Strychinine, a Glycine Inhibitor, on the Programmed Cell Death of Motoneurons during the Chick Development.

In this study, we report that the treatment of strychinine (STR), an inhibitor of glycine receptor, induced premature onset of programmed cell death (PCD) of developing chick motoneurons (MNs). Treatment of STR on E4 chick embryo increased the apoptosis of MN on E5 when MN PCD does not occur normally. On the other hand, treatment of STR from E3 or E5 for 24 hours did not significantly influence the extent of MN PCD, indicating that the STR effect is developmental stage-specific. However, the expression of glycine receptor isoform was low on E3-4, and other glycine receptor antagonists did not exhibit PCD-promoting activity, suggesting that the STR action on PCD is not related to the glycine receptor activation. Identification of the target molecule for STR action may provide novel mechanism how the onset of developmental PCD is regulated.

Expression of connexin29 and 32 in the penumbra region after traumatic brain injury of mice.

Connexins (Cx) are transmembrane proteins forming vertebrate gap junction channels for direct cell-cell communication. We found that the expressions of two Cx family members, Cx29 and Cx32, were progressively increased in the sharp border of injury penumbra regions after cryotraumatic brain injury. Although these two Cxs are expressed exclusively in the oligodendrocytes in the normal cerebral cortex, their expressions were increased in the astrocytes and microglia localized in the injury border. Highly selective induction of Cxs in the injury border suggests that altered Cxs may contribute to the propagations of injury-related and/or regeneration signals after acute brain injury.

Preso, a novel PSD-95-interacting FERM and PDZ domain protein that regulates dendritic spine morphogenesis.

PSD-95 is an abundant postsynaptic density (PSD) protein involved in the formation and regulation of excitatory synapses and dendritic spines, but the underlying mechanisms are not comprehensively understood. Here we report a novel PSD-95-interacting protein Preso that regulates spine morphogenesis. Preso is mainly expressed in the brain and contains WW (domain with two conserved Trp residues), PDZ (PSD-95/Dlg/ZO-1), FERM (4.1, ezrin, radixin, and moesin), and C-terminal PDZ-binding domains. These domains associate with actin filaments, the Rac1/Cdc42 guanine nucleotide exchange factor betaPix, phosphatidylinositol-4,5-bisphosphate, and the postsynaptic scaffolding protein PSD-95, respectively. Preso overexpression increases the density of dendritic spines in a manner requiring WW, PDZ, FERM, and PDZ-binding domains. Conversely, knockdown or dominant-negative inhibition of Preso decreases spine density, excitatory synaptic transmission, and the spine level of filamentous actin. These results suggest that Preso positively regulates spine density through its interaction with the synaptic plasma membrane, actin filaments, PSD-95, and the betaPix-based Rac1 signaling pathway.

Neuroprotective function of thymosin-beta and its derivative peptides on the programmed cell death of chick and rat neurons.

Thymosin-betas (Tbetas) are small polypeptides with various biological functions, including cytoskeletal remodeling, angiogenesis, cellular migration, wound healing, and regulation of apoptosis. Recently, we found that Tbeta is involved in the control of programmed cell death (PCD) of motoneurons (MNs) in chick embryo, and that the anti-apoptotic action of Tbeta is independent of its actin-sequestering activity. In this study, we observed that a synthetic peptide derived from Tbeta suppressed staurosporine-induced neuronal apoptosis in vitro, and PCD of chick or rat MNs in vivo. Furthermore, inhibition of Tbeta4 in chick embryo by antibody significantly augmented the PCD of MNs, suggesting that secreted form of Tbeta is physiological regulator of PCD. Based on these findings, we propose that extracellularly secreted Tbeta is involved in the control of PCD of neurons during development, and Tbeta-derived peptides could be useful for the anti-apoptotic therapy of neuropathologies related to neuronal apoptosis.

Anti-apoptotic function of thymosin-beta in developing chick spinal motoneurons.

Thymosin-betas (Tbetas) are water-soluble peptides abundantly present in the cytoplasm and extracellular compartment. The functions of Tbetas appear to be pleiotrophic, including actin-remodeling, wound healing, angiogenesis, etc. In the present study, we present the evidence that Tbetas have anti-apoptotic activity on developing chick motoneurons (MNs) in vivo. Using in ovo electroporation, we introduced three isoforms of Tbeta (Tbeta4, Tbeta10, and Tbeta15) and found the significantly diminished normal and limb bud removal (LBR)-induced programmed cell death. Such anti-apoptotic activity is independent of Tbeta's actin remodeling activity. On the other hand, overexpression of Tbetas substantially reduced early cell death initiation signal, such as phosphorylation of c-Jun. Collectively, these results suggest that Tbetas may prevent apoptosis of neurons via blockade of early apoptogenic signals independent of actin remodeling action.

Phosphorylation of c-Jun in avian and mammalian motoneurons in vivo during programmed cell death: an early reversible event in the apoptotic cascade.

c-Jun is a transcription factor that is involved in various cellular events, including apoptotic cell death. For example, phosphorylation of c-Jun is one of the earliest biochemical changes detected in dying sympathetic neurons after NGF deprivation in vitro. However, currently, it is not known whether a similar molecular event is involved in the developmental programmed cell death (PCD) of neurons in vivo. We observed that only a subpopulation of motoneurons (MNs) exhibit c-Jun phosphorylation during the PCD period in chick [embryonic day 5 (E5)-E12] and mouse (E13-E18) embryos. Experimental perturbation of MN survival-promoting signals by limb bud removal (reduced signals) or by activity blockade (increased signals) in the chick embryo demonstrated that the presence of those signals is negatively correlated with the number of c-Jun-phosphorylated MNs. This suggests that insufficient survival signals (e.g., neurotrophic factors) may induce c-Jun phosphorylation of MNs in vivo. Consistent with the idea that c-Jun phosphorylation is a reversible event during normal PCD of MNs, we found that c-Jun phosphorylation was transiently observed in a subpopulation of mouse MNs rescued from PCD by deletion of the proapoptotic gene Bax. Inhibition of c-Jun signaling significantly reduced MN death in chick embryo, indicating that activation of c-Jun signaling is necessary for the PCD of MNs. Together, c-Jun phosphorylation appears to be required for the initiation of an early and reversible event in the intracellular PCD cascade in vivo after loss of survival-promoting signals such as neurotrophic factors.