Recruitment of mitochondria into apoptotic signaling correlates with the presence of inclusions formed by amyotrophic lateral sclerosis-associated SOD1 mutations

Mutations in Cu, Zn-superoxide dismutase 1 (SOD1) are associated with degeneration of motor neurons in the disease, familial amyotrophic lateral sclerosis. Intracellular protein inclusions containing mutant SOD1 (mSOD1) are associated with disease but it is unclear whether they are neuroprotective or cytotoxic. We report here that the formation of mSOD1 inclusions in a motor neuron-like cell line (NSC-34) strongly correlates with apoptosis via the mitochondrial death pathway. Applying confocal microscopic analyses, we observed changes in nuclear morphology and activation of caspase 3 specifically in cells expressing mSOD1 A4V or G85R inclusions. Furthermore, markers of mitochondrial apoptosis (activation and recruitment of Bax, and cytochrome c redistribution) were observed in 30% of cells bearing mSOD1 inclusions but not in cells expressing dispersed SOD1. In the presence of additional apoptotic challenges (staurosporine, etoposide, and hydrogen peroxide), cells bearing mSOD1 inclusions were susceptible to further apoptosis suggesting they were in a pro-apoptotic state, thus confirming that inclusions are linked to toxicity. Surprisingly, cells displaying dispersed SOD1 [both wildtype (WT) and mutant] were protected against apoptosis upstream of mitochondrial apoptotic signaling, induced by all agents tested. This protection against apoptosis was unrelated to SOD1 enzymatic activity because the G85R that lacks enzymatic function protected cells similarly to both WT SOD1 and A4V that possesses WT-like activity. These findings demonstrate new aspects of SOD1 in relation to cellular viability; specifically, mSOD1 can be either neuroprotective or cytotoxic depending on its aggregation state.     

A metabolic switch in brain: glucose and lactate metabolism modulation by ascorbic acid

In this review, we discuss a novel function of ascorbic acid in brain energetics. It has been proposed that during glutamatergic synaptic activity neurons preferably consume lactate released from glia. The key to this energetic coupling is the metabolic activation that occurs in astrocytes by glutamate and an increase in extracellular [K⁺]. Neurons are cells well equipped to consume glucose because they express glucose transporters and glycolytic and tricarboxylic acid cycle enzymes. Moreover, neuronal cells express monocarboxylate transporters and lactate dehydrogenase isoenzyme 1, which is inhibited by pyruvate. As glycolysis produces an increase in pyruvate concentration and a decrease in NAD⁺/NADH, lactate and glucose consumption are not viable at the same time. In this context, we discuss ascorbic acid participation as a metabolic switch modulating neuronal metabolism between rest and activation periods. Ascorbic acid is highly concentrated in CNS. Glutamate stimulates ascorbic acid release from astrocytes. Ascorbic acid entry into neurons and within the cell can inhibit glucose consumption and stimulate lactate transport. For this switch to occur, an ascorbic acid flow is necessary between astrocytes and neurons, which is driven by neural activity and is part of vitamin C recycling. Here, we review the role of glucose and lactate as metabolic substrates and the modulation of neuronal metabolism by ascorbic acid.     

Topographic cues of nano-scale height direct neuronal growth pattern

We study the role of nano-scale cues in controlling neuronal growth. We use photolithography to fabricate substrates with repeatable line-pattern ridges of nano-scale heights. We find that neuronal processes, which are of micron size, have strong interactions with ridges even as low as 10 nm. The interaction between the neuronal process and the ridge leads to a deflection of growth direction and a preferred alignment with the ridges. The interaction strength clearly depends on the ridges' height. For 25 nm ridges approximately half of the neuronal processes are modified, while at 100 nm the majority of neurites change their original growth direction post interaction. In addition, the effect on growth correlates with the incoming angle between the neuronal process and the ridge. We underline the adhesion as a key mechanism in directing neuronal growth. Our study highlights the sensitivity of growing neurites to nano-scale cues thus opens a new avenue of research for pre-designed neuronal growth and circuitry.     

Glypican-1, phosphacan/receptor protein-tyrosine phosphatase-ζ/β and its ligand,tenascin-C,are expressed by neural stem cells and neural cells derived from embryonic stem cells

The heparan sulfate proteoglycan glypican-1, the chondroitin sulfate proteoglycan phosphacan/RPTP (receptor protein-tyrosine phosphatase)-ζ/β and the extracellular matrix protein tenascin-C were all found to be expressed by neural stem cells and by neural cells derived from them. Expression of proteoglycans and tenascin-C increased after retinoic acid induction of SSEA1-positive ES (embryonic stem) cells to nestin-positive neural stem cells, and after neural differentiation, proteoglycans and tenascin-C are expressed by both neurons and astrocytes, where they surround cell bodies and processes and in certain cases show distinctive expression patterns. With the exception of tenascin-C (whose expression may decrease somewhat), expression levels do not change noticeably during the following 2 weeks in culture. The significant expression, by neural stem cells and neurons and astrocytes derived from them, of two major heparan sulfate and chondroitin sulfate proteoglycans of nervous tissue and of tenascin-C, a high-affinity ligand of phosphacan/RPTP-ζ/β, indicates that an understanding of their specific functional roles in stem cell neurobiology will be important for the therapeutic application of this new technology in facilitating nervous tissue repair and regeneration.     

Immunonucleochemistry: a new method for in situ detection of antigens in the nucleus of cells in culture

The advancement of immunocytochemistry (ICC) allows one to observe detailed spatial distribution of cellular antigens, but, with some limitations. Using conventional ICC, it is difficult to distinguish the nuclear localization from cytoplasm, as two large subcellular compartments overlap on the z-axis. In this study, we have investigated whether in situ immunostaining of ‘naked’ nuclei could provide an unambiguous method for detection of nuclear antigens. We have designed a protocol that efficiently lyses plasmalemma, while keeping the nuclear envelope intact. The optimal condition for lysing the plasmalemma was 0.5% Nonidet P-40 for 5 min in both neuronal and non-neuronal cultured cells. Using this protocol, we could unambiguously isolate nuclear from cytoplasmic ICC signals. Since the present protocol has been designed for immunostaining of ‘naked’ nuclei from cultured or isolated cells, we have coined a new term to refer to this procedure as ‘immunonucleochemistry’ (‘INC’ for abbreviation).     

A trade-off switch of two immunological memories in Caenorhabditis elegans reinfected by bacterial pathogens

Recent studies have suggested that innate immune responses exhibit characteristics associated with memory linked to modulations in both vertebrates and invertebrates. However, the diverse evolutionary paths taken, particularly within the invertebrate taxa, should lead to similarly diverse innate immunity memory processes. Our understanding of innate immune memory in invertebrates primarily comes from studies of the fruit fly Drosophila melanogaster, the generality of which is unclear. Caenorhabditis elegans typically inhabits soil harboring a variety of fatal microbial pathogens; for this invertebrate, the innate immune system and aversive behavior are the major defensive strategies against microbial infection. However, their characteristics of immunological memory remains infantile. Here we discovered an immunological memory that promoted avoidance and suppressed innate immunity during reinfection with bacteria, which we revealed to be specific to the previously exposed pathogens. During this trade-off switch of avoidance and innate immunity, the chemosensory neurons AWB and ADF modulated production of serotonin and dopamine, which in turn decreased expression of the innate immunity-associated genes and led to enhanced avoidance via the downstream insulin-like pathway. Therefore, our current study profiles the immune memories during C. elegans reinfected by pathogenic bacteria and further reveals that the chemosensory neurons, the neurotransmitter(s), and their associated molecular signaling pathways are responsible for a trade-off switch between the two immunological memories.     

miR-22-3p enhances the intrinsic regenerative abilities of primary sensory neurons via the CBL/p-EGFR/p-STAT3/GAP43/p-GAP43 axis

Spinal cord injury (SCI) is a devastating disease. Strategies that enhance the intrinsic regenerative ability are very important for the recovery of SCI to radically prevent the occurrence of sensory disorders. Epidermal growth factor (EGF) showed a limited effect on the growth of primary sensory neuron neurites due to the degradation of phosphorylated-epidermal growth factor receptor (p-EGFR) in a manner dependent on Casitas B-lineage lymphoma (CBL) (an E3 ubiquitin-protein ligase). MiR-22-3p predicted from four databases could target CBL to inhibit the expression of CBL, increase p-EGFR levels and neurites length via STAT3/GAP43 pathway rather than Erk1/2 axis. EGF, EGFR, and miR-22-3p were downregulated sharply after injury. In vivo miR-22-3p Agomir application could regulate CBL/p-EGFR/p-STAT3/GAP43/p-GAP43 axis, and restore spinal cord sensory conductive function. This study clarified the mechanism of the limited promotion effect of EGF on adult primary sensory neuron neurite and targeting miR-22-3p could be a novel strategy to treat sensory dysfunction after SCI.