Upregulating Lin28a Promotes Axon Regeneration in Adult Mice with Optic Nerve and Spinal Cord Injury.

Severed CNS axons fail to regenerate in adult mammals and there are no effective regenerative strategies to treat patients with CNS injuries. Several genes, including phosphatase and tensin homolog (PTEN) and Krüppel-like factors, regulate intrinsic growth capacity of mature neurons. The Lin28 gene is essential for cell development and pluripotency in worms and mammals. In this study, we evaluated the role of Lin28a in regulating regenerative capacity of diverse populations of CNS neurons in adult mammals. Using a neuron-specific Thy1 promoter, we generated transgenic mice that overexpress Lin28a protein in multiple populations of projection neurons, including corticospinal tracts and retinal ganglion cells. We demonstrate that upregulation of Lin28a in transgenic mice induces significant long distance regeneration of both corticospinal axons and the optic nerve in adult mice. Importantly, overexpression of Lin28a by post-injury treatment with adeno-associated virus type 2 (AAV2) vector stimulates dramatic regeneration of descending spinal tracts and optic nerve axons after lesions. Upregulation of Lin28a also enhances activity of the Akt signaling pathway in mature CNS neurons. Therefore, Lin28a is critical for regulating growth capacity of multiple CNS neurons and may become an important molecular target for treating CNS injuries.

Promoting Axon Regeneration in Adult CNS by Targeting Liver Kinase B1.

Liver kinase B1 (LKB1), a downstream effector of cyclic AMP (cAMP)/PKA and phosphatidylinositol 3-kinase (PI3K) pathways, is a determinant for migration and differentiation of many cells, but its role in CNS axon regeneration is unknown. Therefore, LKB1 was overexpressed in sensorimotor cortex of adult mice five days after mid-thoracic spinal cord injury, using an AAV2 vector. Regeneration of corticospinal axons was dramatically enhanced. Next, systemic injection of a mutant-AAV9 vector was used to upregulate LKB1 specifically in neurons. This promoted long-distance regeneration of injured corticospinal fibers into caudal spinal cord in adult mice and regrowth of descending serotonergic and tyrosine hydroxylase immunoreactive axons. Either intracortical or systemic viral delivery of LKB1 significantly improved recovery of locomotor functions in adult mice with spinal cord injury. Moreover, we demonstrated that LKB1 used AMPKα, NUAK1, and ERK as the downstream effectors in the cortex of adult mice. Thus, LKB1 may be a critical factor for enhancing the growth capacity of mature neurons and may be an important molecular target in the treatment of CNS injuries.

Stabilization of Microtubule-Unbound Tau via Tau Phosphorylation at Ser262/356 by Par-1/MARK Contributes to Augmentation of AD-Related Phosphorylation and Aß42-Induced Tau Toxicity.

Abnormal accumulation of the microtubule-interacting protein tau is associated with neurodegenerative diseases including Alzheimer's disease (AD). ß-amyloid (Aß) lies upstream of abnormal tau behavior, including detachment from microtubules, phosphorylation at several disease-specific sites, and self-aggregation into toxic tau species in AD brains. To prevent the cascade of events leading to neurodegeneration in AD, it is essential to elucidate the mechanisms underlying the initial events of tau mismetabolism. Currently, however, these mechanisms remain unclear. In this study, using transgenic Drosophila co-expressing human tau and Aß, we found that tau phosphorylation at AD-related Ser262/356 stabilized microtubule-unbound tau in the early phase of tau mismetabolism, leading to neurodegeneration. Aß increased the level of tau detached from microtubules, independent of the phosphorylation status at GSK3-targeted SP/TP sites. Such mislocalized tau proteins, especially the less phosphorylated species, were stabilized by phosphorylation at Ser262/356 via PAR-1/MARK. Levels of Ser262 phosphorylation were increased by Aß42, and blocking this stabilization of tau suppressed Aß42-mediated augmentation of tau toxicity and an increase in the levels of tau phosphorylation at the SP/TP site Thr231, suggesting that this process may be involved in AD pathogenesis. In contrast to PAR-1/MARK, blocking tau phosphorylation at SP/TP sites by knockdown of Sgg/GSK3 did not reduce tau levels, suppress tau mislocalization to the cytosol, or diminish Aß-mediated augmentation of tau toxicity. These results suggest that stabilization of microtubule-unbound tau by phosphorylation at Ser262/356 via the PAR-1/MARK may act in the initial steps of tau mismetabolism in AD pathogenesis, and that such tau species may represent a potential therapeutic target for AD.

Neuronal activity-dependent local activation of dendritic unfolded protein response promotes expression of brain-derived neurotrophic factor in cell soma.

Unfolded protein response (UPR) has roles not only in resolving the accumulation of unfolded proteins owing to endoplasmic reticulum (ER) stress, but also in regulation of cellular physiological functions. ER stress transducers providing the branches of UPR signaling are known to localize in distal dendritic ER of neurons. These reports suggest that local activation of UPR branches may produce integrated outputs for distant communication, and allow regulation of local events in highly polarized neurons. Here, we demonstrated that synaptic activity- and brain-derived neurotrophic factor (BDNF)-dependent local activation of UPR signaling could be associated with dendritic functions through retrograde signal propagation by using murine neuroblastoma cell line, Neuro-2A and primary cultured hippocampal neurons derived from postnatal day 0 litter C57BL/6 mice. ER stress transducer, inositol-requiring kinase 1 (IRE1), was activated at postsynapses in response to excitatory synaptic activation. Activated dendritic IRE1 accelerated accumulation of the downstream transcription factor, x-box-binding protein 1 (XBP1), in the nucleus. Interestingly, excitatory synaptic activation-dependent up-regulation of XBP1 directly facilitated transcriptional activation of BDNF. BDNF in turn drove its own expression via IRE1-XBP1 pathway in a protein kinase A-dependent manner. Exogenous treatment with BDNF promoted extension and branching of dendrites through the protein kinase A-IRE1-XBP1 cascade. Taken together, our findings indicate novel mechanisms for communication between soma and distal sites of polarized neurons that are coordinated by local activation of IRE1-XBP1 signaling. Synaptic activity- and BDNF-dependent distinct activation of dendritic IRE1-XBP1 cascade drives BDNF expression in cell soma and may be involved in dendritic extension. Cover Image for this issue: doi. 10.1111/jnc.14159.

Protein tyrosine phosphatase σ regulates autoimmune encephalomyelitis development.

Protein tyrosine phosphatases (PTPs) play essential roles in regulating signaling events in multiple cells by tyrosine dephosphorylation. One of them, PTPσ, appears important in regulating function of plasmacytoid dendritic cells (pDC). Here we report that PTPσ deletion in knockout mice and inhibition with a selective antagonist peptide exacerbated symptoms of experimental autoimmune encephalomyelitis (EAE) by enhancing axon and myelin damage in the spinal cord. PTPσ-/- mice displayed pro-inflammatory profiles in the spinal cord and lymphoid organs following MOG peptide immunization. PTPσ deletion promoted a pro-inflammatory phenotype in conventional DCs and directly regulated differentiation of CD4+ T cells. It also facilitated infiltration of T lymphocytes, activation of macrophages in the CNS and development of EAE. Therefore, PTPσ is a key negative regulator in EAE initiation and progression, which acts by regulating functions of DCs, T cells, and other immune cells. PTPσ may become an important molecular target for treating autoimmune disorders.

ER Stress and Disease: Toward Prevention and Treatment.

Secretory and membrane proteins are synthesized in ribosomes, then mature in the endoplasmic reticulum (ER), but if ER function is impaired, immature defective proteins accumulate in the ER. This situation is called ER stress: in response, a defensive mechanism called the unfolded protein response (UPR) is activated in cells to reduce the defective proteins. During the UPR, the ER transmembrane sensor molecules inositol-requiring enzyme 1 (IRE1), activating transcription factor 6 (ATF6), and RNA-dependent protein kinase (PKR)-like ER kinase (PERK) are activated, stress signals are transduced to the outside of the ER, and various cell responses, including gene induction, occur. In ER-associated degradation (ERAD), one type of UPR, defective proteins are eventually expelled from the ER and degraded in the cytoplasm through the ubiquitin proteasome system. Since ER stress has been reported to have relationships with neurodegenerative diseases, diabetes, metabolic syndromes, and cancer, it is the focus of increased attention from the perspectives of elucidating pathogenic mechanisms, and in the development of therapeutics.

Tau phosphorylation at Alzheimer's disease-related Ser356 contributes to tau stabilization when PAR-1/MARK activity is elevated.

Abnormal phosphorylation of the microtubule-associated protein tau is observed in many neurodegenerative diseases, including Alzheimer's disease (AD). AD-related phosphorylation of two tau residues, Ser262 and Ser356, by PAR-1/MARK stabilizes tau in the initial phase of mismetabolism, leading to subsequent phosphorylation events, accumulation, and toxicity. However, the relative contribution of phosphorylation at each of these sites to tau stabilization has not yet been elucidated. In a Drosophila model of human tau toxicity, we found that tau was phosphorylated at Ser262, but not at Ser356, and that blocking Ser262 phosphorylation decreased total tau levels. By contrast, when PAR-1 was co-overexpressed with tau, tau was hyperphosphorylated at both Ser262 and Ser356. Under these conditions, the protein levels of tau were significantly elevated, and prevention of tau phosphorylation at both residues was necessary to completely suppress this elevation. These results suggest that tau phosphorylation at Ser262 plays the predominant role in tau stabilization when PAR-1/MARK activity is normal, whereas Ser356 phosphorylation begins to contribute to this process when PAR-1/MARK activity is abnormally elevated, as in diseased brains.

Role of CSPG receptor LAR phosphatase in restricting axon regeneration after CNS injury.

Extracellular matrix molecule chondroitin sulfate proteoglycans (CSPGs) are highly upregulated in scar tissues and form a potent chemical barrier for CNS axon regeneration. Recent studies support that the receptor protein tyrosine phosphatase σ (PTPσ) and its subfamily member leukocyte common antigen related phosphatase (LAR) act as transmembrane receptors to mediate CSPG inhibition. PTPσ deficiency increased regrowth of ascending axons into scar tissues and descending corticospinal tract (CST) axons into the caudal spinal cord after spinal cord injury (SCI). Pharmacological LAR inhibition enhanced serotonergic axon growth in SCI mice. However, transgenic LAR deletion on axon growth in vivo and the role of LAR in regulating regrowth of other fiber tracts have not been studied. Here, we studied the role of LAR in restricting regrowth of injured descending CNS axons in deficient mice. LAR deletion increased regrowth of serotonergic axons into scar tissues and caudal spinal cord after dorsal over-hemitransection. LAR deletion also stimulated regrowth of CST fibers into the caudal spinal cord. LAR protein was upregulated days to weeks after injury and co-localized to serotonergic and CST axons. Moreover, LAR deletion improved functional recovery by increasing BMS locomotor scores and stride length and reducing grid walk errors. This is the first transgenic study that demonstrates the crucial role of LAR in restricting regrowth of injured CNS axons.

Loss of axonal mitochondria promotes tau-mediated neurodegeneration and Alzheimer's disease-related tau phosphorylation via PAR-1.

Abnormal phosphorylation and toxicity of a microtubule-associated protein tau are involved in the pathogenesis of Alzheimer's disease (AD); however, what pathological conditions trigger tau abnormality in AD is not fully understood. A reduction in the number of mitochondria in the axon has been implicated in AD. In this study, we investigated whether and how loss of axonal mitochondria promotes tau phosphorylation and toxicity in vivo. Using transgenic Drosophila expressing human tau, we found that RNAi-mediated knockdown of milton or Miro, an adaptor protein essential for axonal transport of mitochondria, enhanced human tau-induced neurodegeneration. Tau phosphorylation at an AD-related site Ser262 increased with knockdown of milton or Miro; and partitioning defective-1 (PAR-1), the Drosophila homolog of mammalian microtubule affinity-regulating kinase, mediated this increase of tau phosphorylation. Tau phosphorylation at Ser262 has been reported to promote tau detachment from microtubules, and we found that the levels of microtubule-unbound free tau increased by milton knockdown. Blocking tau phosphorylation at Ser262 site by PAR-1 knockdown or by mutating the Ser262 site to unphosphorylatable alanine suppressed the enhancement of tau-induced neurodegeneration caused by milton knockdown. Furthermore, knockdown of milton or Miro increased the levels of active PAR-1. These results suggest that an increase in tau phosphorylation at Ser262 through PAR-1 contributes to tau-mediated neurodegeneration under a pathological condition in which axonal mitochondria is depleted. Intriguingly, we found that knockdown of milton or Miro alone caused late-onset neurodegeneration in the fly brain, and this neurodegeneration could be suppressed by knockdown of Drosophila tau or PAR-1. Our results suggest that loss of axonal mitochondria may play an important role in tau phosphorylation and toxicity in the pathogenesis of AD.

Glial senescence enhances α-synuclein pathology owing to its insufficient clearance caused by autophagy dysfunction.

Parkinson's disease (PD) is characterized by the pathological accumulation of α-synuclein (α-syn) and loss of dopaminergic neurons in the substantia nigra. Aging is a significant risk factor for PD. The accumulation of senescent glial cells in the aged brain contributes to PD progression by inducing chronic neuroinflammatory processes. However, although the insufficient degradation of α-syn aggregates results in PD deterioration, the possible alteration in the ability of α-syn clearance in senescent glia has received little attention. In this study, we investigated how aging and glial senescence affect the capacity of α-syn clearance. We found that following the intra-striatal injection of human α-syn (hu-α-syn) preformed fibril, hu-α-syn pathology persisted more in aged mice compared with younger mice and that aged microglia exhibited greater accumulation of hu-α-syn than younger microglia. Moreover, in vitro assay revealed that the clearance of hu-α-syn was primarily dependent on the autophagy-lysosome system rather than on the ubiquitin-proteasome system and that the capacity of hu-α-syn clearance was diminished in senescent glia because of autophagy-lysosome system dysfunction. Overall, this study provides new insights into the role of senescent glia in PD pathogenesis.

Effect of aging on norepinephrine-related proliferative response in primary cultured periportal and perivenous hepatocytes.

Norepinephrine (NE) amplifies the mitogenic effect of EGF in a rat liver through the adrenergic receptor coupled with G protein, Ghα. Ghα is also known as a transglutaminase 2 (TG2), whose cross-linking activity is implicated in hepatocyte growth. Recently, we found that NE-induced amplification of EGF-induced DNA synthesis in hepatocytes obtained from perivenous regions of liver is caused by inhibiting the downregulation of EGF receptor (EGFR) by TG2. In the present study, we investigated the effect of aging on NE-related proliferative response. Hepatocytes were obtained from the liver of 7- and 90-wk-old rats. To examine this in detail, periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated using the digitonin/collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, G protein activity, and TG2 activity were measured. NE slightly potentiated [125I]EGF binding to EGFR, and EGF-induced DNA synthesis in PVH but not in PPH. [3H]NE binding studies indicated that PVH have a greater number of receptors than PPH, and that the number of receptors in both subpopulations increased with aging. NE-induced changes in G protein activity and TG2 activity in 90-wk-old rats were slight compared with 7-wk-old rats. These results suggest that NE results in a slight recovery effect on the age-related decline in EGF-induced DNA synthesis because of incomplete switching of the function from TG2 to Ghα.

Norepinephrine modulates the zonally different hepatocyte proliferation through the regulation of transglutaminase activity.

A neurotransmitter, norepinephrine (NE), amplifies the mitogenic effect of epidermal growth factor (EGF) in the liver by acting on the alpha(1)-adrenergic receptor coupled with G protein, Galpha(h). However, the molecular mechanism is not well understood. Galpha(h) is known as a transglutaminase 2 (TG2), a cross-linking enzyme implicated in hepatocyte proliferation. We investigated the effect of NE on EGF-induced cell proliferation and TG2 activity using hepatocytes isolated in periportal and perivenous regions of the liver, which differ in proliferative capacity. Periportal hepatocytes (PPH) and perivenous hepatocytes (PVH) were isolated by the digitonin-collagenase perfusion technique. EGF or NE receptor binding was analyzed by Scatchard analysis. Changes in NE-induced DNA synthesis, EGF receptor (EGFR) dimerization and phosphorylation, and TG2 activity were measured. NE enhanced EGF-induced DNA synthesis, EGF-induced EGFR dimerization, and its phosphorylation in PVH but not in PPH. [(3)H]NE binding studies indicated that PVH was found to have a greater affinity and number of receptors than PPH. Furthermore, NE treatment decreased TG2 activity and increased phospholipase C activity in PVH although TG2 level showed no change. These results suggest that NE-induced amplification of EGF-induced DNA synthesis especially in PVH is caused by upregulation of EGFR activation through the switching of function from TG2 to Galpha(h).

Immunotherapies in Huntington's disease and α-Synucleinopathies.

Modulation of immune activation using immunotherapy has attracted considerable attention for many years as a potential therapeutic intervention for several inflammation-associated neurodegenerative diseases. However, the efficacy of single-target immunotherapy intervention has shown limited or no efficacy in alleviating disease burden and restoring functional capacity. Marked immune system activation and neuroinflammation are important features and prodromal signs in polyQ repeat disorders and α-synucleinopathies. This review describes the current status and future directions of immunotherapies in proteinopathy-induced neurodegeneration with emphasis on preclinical and clinical efficacies of several anti-inflammatory compounds and antibody-based therapies for the treatment of Huntington's disease and α-synucleinopathies. The review concludes with how disease modification and functional restoration could be achieved by using targeted multimodality therapy to target multiple factors.

Stabilities of 67ga- and 111In-labeled transferrin in vitro.

We attempted to develop a stable radiolabeled transferrin (Tf) useful in experimental studies related to Tf receptor. 67Ga and 111In were used as labeling radioisotopes. The results from gel chromatography, dialysis, and electrophoresis showed that 111In-DTPA-Tf was the most stable among the radiolabeled Tfs examined in the present study. 111In-DTPA-Tf was also the most stable radiolabeled transferrin in the blood.

Effect of retinoic acid on transglutaminase and ornithine decarboxylase activities during liver regeneration.

Liver regeneration is regulated by several factors, including growth factors, cytokines, and post-translational modifications of several proteins. It is suggested that transglutaminase 2 (TG2) and ornithine decarboxylase (ODC) are involved in liver regeneration. To investigate the role of TG2 and ODC activities in regenerating liver, we used retinoic acid (RA), an inducer of TG2 and a suppressor of ODC. Regenerating rat liver was prepared by 70% partial hepatectomy (PH). Rats were sacrificed at 1, 2, 3, 4, and 6 days after surgery. RA was intraperitoneally injected immediately after PH. TG2 and ODC activities and products (epsilon-(gamma-glutamyl) lysine isopeptide (Gln-Lys) and polyamines, respectively) were examined at the indicated times. In RA-treated rat, DNA synthesis and ODC activity declined and the peak shifted to 2 days after PH, whereas TG2 activity increased at 1 day after PH. At that time, protein-polyamine, especially the protein-spermidine (SPD) bond, transiently decreased, whereas the formation of the Gln-Lys bond increased after PH. These results suggested that in regenerating liver, enhanced the formation of Gln-Lys bonds catalyzed by TG2 led to reduced DNA synthesis, whereas when ODC produced newly synthesized SPD, the inhibition of Gln-Lys bond production by the preferential formation of protein-SPD bonds led to an increase in DNA synthesis.

The uptake of 111In in the liver and bone marrow of partially hepatectomized and venesectioned rats.

Indium-111 ((111)In) has a strong binding affinity for transferrin (Tf), and the (111)In-Tf complex binds to Tf receptor in various tissues. In partial hepatectomy (PH), a part of blood in circulation is lost along with removed liver tissues; consequently, the number of blood cells and the amount of Tf in circulation decreases. These decreases should greatly affect the uptake of (111)In in the liver and bone marrow. In order to investigate this effect, we compared the uptake in partially hepatectomized rats with that in venesectioned rats, in which only the volume of blood in circulation had been decreased. Our data show that fewer blood cells and smaller amount of Tf in circulation due to venesection increased the uptake of (111)In in bone marrow, but not in the liver, whereas PH increased the uptake of (111)In in both bone marrow and liver. The higher bone marrow uptake of (111)In must be related to the increase of the hematopoietic function resulted from the smaller amount of blood; the higher uptake in liver may be closely related to liver regeneration.

Diverse functions of protein tyrosine phosphatase σ in the nervous and immune systems.

Tyrosine phosphorylation is a common means of regulating protein functions and signal transduction in multiple cells. Protein tyrosine phosphatases (PTPs) are a large family of signaling enzymes that remove phosphate groups from tyrosine residues of target proteins and change their functions. Among them, receptor-type PTPs (RPTPs) exhibit a distinct spatial pattern of expression and play essential roles in regulating neurite outgrowth, axon guidance, and synaptic organization in developmental nervous system. Some RPTPs function as essential receptors for chondroitin sulfate proteoglycans that inhibit axon regeneration following CNS injury. Interestingly, certain RPTPs are also important to regulate functions of immune cells and development of autoimmune diseases. PTPσ, a RPTP in the LAR subfamily, is expressed in various immune cells and regulates their differentiation, production of various cytokines and immune responses. In this review, we highlight the physiological and pathological significance of PTPσ and related molecules in both nervous and immune systems.

Axonal Activation of the Unfolded Protein Response Promotes Axonal Regeneration Following Peripheral Nerve Injury.

Adult mammalian peripheral neurons have an intrinsic regrowth capacity in response to axonal injury. The induction of calcium ion (Ca2+) oscillations at an injured site is critical for the regulation of regenerative responses. In polarized neurons, distal axonal segments contain a well-developed endoplasmic reticulum (ER) network that is responsible for Ca2+ homeostasis. Although these characteristics implicate the relevance among injury-induced Ca2+ dynamics, axonal ER-derived signaling, and regenerative responses propagated along the axons, the details are not fully understood. In the present study, we found that Ca2+ release from the axonal ER was accelerated in response to injury. Additionally, axonal injury-dependent Ca2+ release from the ER activated unfolded protein response (UPR) signaling at injured sites. Inhibition of axonal UPR signaling led to fragmentation of the axonal ER and disrupted growth cone formation, suggesting that activation of axonal UPR branches following axonal injury promotes regeneration via regulation of ER reconstruction and formation of growth cones. Our studies revealed that local activation of axonal UPR signaling by injury-induced Ca2+ release from the ER is critical for regeneration. These findings provide a new concept for the link between injury-induced signaling at a distant location and regulation of organelle and cytoskeletal formation in the orchestration of axonal regeneration.

S6K/p70S6K1 protects against tau-mediated neurodegeneration by decreasing the level of tau phosphorylated at Ser262 in a Drosophila model of tauopathy.

Abnormal accumulation of the microtubule-associated protein tau is thought to cause neuronal cell death in a group of age-associated neurodegenerative disorders. Tau is phosphorylated at multiple sites in diseased brains, and phosphorylation of tau at Ser262 initiates tau accumulation and toxicity. In this study, we sought to identify novel factors that affect the metabolism and toxicity of tau phosphorylated at Ser262 (pSer262-tau). A biased screen using a Drosophila model of tau toxicity revealed that knockdown of S6K, the Drosophila homolog of p70S6K1, increased the level of pSer262-tau and enhanced tau toxicity. S6K can be activated by the insulin signaling, however, unlike knockdown of S6K, knockdown of insulin receptor or insulin receptor substrate nonselectively decreased total tau levels via autophagy. Importantly, activation of S6K significantly suppressed tau-mediated axon degeneration, whereas manipulation of either the insulin signaling pathway or autophagy did not. Our results suggest that activation of S6K may be an effective therapeutic strategy for selectively decreasing the levels of toxic tau species and suppressing neurodegeneration.

Sec16A, a key protein in COPII vesicle formation, regulates the stability and localization of the novel ubiquitin ligase RNF183.

We identified 37 ubiquitin ligases containing RING-finger and transmembrane domains. Of these, we found that RNF183 is abundantly expressed in the kidney. RNF183 predominantly localizes to the endoplasmic reticulum (ER), Golgi, and lysosome. We identified Sec16A, which is involved in coat protein complex II vesicle formation, as an RNF183-interacting protein. RNF183 colocalized with Sec16A and interacted through the central conserved domain (CCD) of Sec16A. Although Sec16A is not a substrate for RNF183, RNF183 was more rapidly degraded by the ER-associated degradation (ERAD) in the absence of Sec16A. Sec16A also stabilized the interacting ubiquitin ligase RNF152, which localizes to the lysosome and has structural similarity with RNF183. These results suggest that Sec16A appears to regulate the protein stability and localization of lysosomal ubiquitin ligases.