Norihiro Suzuki

Ambivalent aspects of interleukin-6 in cerebral ischemia: inflammatory versus neurotrophic aspects

Interleukin-6 (IL-6) is pleiotropic cytokine involved in many central nervous system disorders including stroke, and elevated serum IL-6 has been found in acute stroke patients. IL-6 is implicated in the inflammation, which contributes to both injury and repair process after cerebral ischemia. However, IL-6 is one of the neurotrophic cytokines sharing a common receptor subunit, gp130, with other neurotrophic cytokines, such as leukemia inhibitory factor (LIF) and ciliary neurotrophic factor. The expression of IL-6 is most prominently identified in neurons in the peri-ischemic regions, and LIF expression shows a similar pattern. The direct injection of these cytokines into the brain after ischemia can reduce ischemic brain injury. The cytokine receptors are localized on the neuron surface, suggesting that neurons are the cytokine target. The major IL-6 downstream signaling pathway is JAK—STAT, and Stat3 activation occurs mainly in neurons during postischemic reperfusion. Further investigation is necessary to clarify the exact role of Stat3 signaling in neuroprotection. Taken together, the information suggests that IL-6 plays a double role in cerebral ischemia, as an inflammatory mediator during the acute phase and as a neurotrophic mediator between the subacute and prolonged phases.

Characterization of alternative isoforms and inclusion body of the TAR DNA-binding protein-43

TAR DNA-binding protein-43 (TDP-43) has been recently identified as a major component of the ubiquitinated inclusions found in frontotemporal lobar degeneration with ubiquitin-positive inclusions and in amyotrophic lateral sclerosis, diseases that are collectively termed TDP-43 proteinopathies. Several amyotrophic lateral sclerosis-linked mutations of the TDP-43 gene have also been identified; however, the precise molecular mechanisms underlying the neurodegeneration remain unclear. To investigate the biochemical characteristics of TDP-43, we examined truncation, isoforms, and cytoplasmic inclusion (foci) formation using TDP-43-expressing cells. Under apoptosis, caspase-3 generates two 35-kDa (p35f) and 25-kDa (p25f) fragments. However, in caspase-3(−/−) cells, novel caspase-3-independent isoforms of these two variants (p35iso and p25iso) were also detected under normal conditions. With a deletion mutant series, the critical domains for generating both isoforms were determined and applied to in vitro transcription/translation, revealing alternate in-frame translation start sites downstream of the natural initiation codon. Subcellular localization analysis indicated that p35 (p35f and p35iso) expression leads to the formation of stress granules, cellular structures that package mRNA and RNA-binding proteins during cell stress. After applying proteasome inhibitors, aggresomes, which are aggregates of misfolded proteins, were formed in the cytoplasm of cells expressing p35. Collectively, this study demonstrates that the 35-kDa isoforms of TDP-43 assemble in stress granules, suggesting that TDP-43 plays an important role in translation, stability, and metabolism of mRNA. Our findings provide new biological and pathological insight into the development of TDP-43 proteinopathies.

Seipinopathy: a novel endoplasmic reticulum stress-associated disease

The Seipin/BSCL2 gene was originally identified as a loss-of-function gene for congenital generalized lipodystrophy type 2 (CGL2), a condition characterized by severe lipoatrophy, insulin resistance, hypertriglyceridaemia and mental retardation. Recently, gain-of-toxic-function mutations (namely, mutations N88S and S90L) in the seipin gene have been identified in autosomal dominant motor neuron diseases such as Silver syndrome/spastic paraplegia 17 (SPG17) (OMIM #270685) and distal hereditary motor neuropathy type V (dHMN-V) (OMIM #182960). Detailed phenotypic analyses have revealed that upper motor neurons, lower motor neurons and peripheral motor axons are variously affected in patients with these mutations. The clinical spectrum for these mutations is broad, encompassing Silver syndrome, some variants of Charcot-Marie-Tooth disease type 2, dHMNV and spastic paraplegia, even within a common pedigree. Therefore, we propose that seipin-related motor neuron diseases can be collectively referred to as seipinopathies. Expression of the seipin protein can be detected in motor neurons in the spinal cord and white matter in the frontal lobe. This is consistent with the distribution of seipinopathies in the upper and lower motor neurons. Recent studies have shown that seipin, an endoplasmic reticulum (ER)-resident membrane protein, is an N-glycosylated protein that is proteolytically cleaved into N- and C-terminal fragments and is polyubiquitinated. Interestingly, the N88S and S90L mutations are in the N-glycosylation motif, and these mutations enhance ubiquitination and degradation of seipin by the ubiquitin-proteasome system (UPS). Furthermore, both mutations appear to result in proteins that are improperly folded, which leads to accumulation of the mutant protein in the ER. We have shown that expression of mutant forms of seipin in cultured cells activates the unfolded protein response (UPR) pathway and induces ER stress-mediated cell death. These findings suggest that seipinopathies are novel conformational diseases and that neurodegeneration in these diseases is tightly associated with ER stress, which has recently been reported to be associated with other neurodegenerative diseases. Further study of the pathological mechanisms of the mutant forms of seipin may lead to important new insights into motor neuron diseases, including other spastic paraplegia diseases and amyotrophic lateral sclerosis.

Regeneration of the central nervous system using endogenous repair mechanisms.

Recent advances in developmental and stem cell biology have made regeneration‐based therapies feasible as therapeutic strategies for patients with damaged central nervous systems (CNSs), including those with spinal cord injuries, Parkinson disease, or stroke. These strategies can be classified into two approaches: (i) the replenishment of lost neural cells and (ii) the induction of axonal regeneration. The first approach includes the activation of endogenous neural stem cells (NSCs) in the adult CNS and cell transplantation therapy. Endogenous NSCs have been shown to give rise to new neurons after insults, including ischemia, have been sustained; this form of neurogenesis followed by the migration and functional maturation of neuronal cells, as well as the responses of glial cells and the vascular system play crucial roles in endogenous repair mechanisms in damaged CNS tissue. In this review, we will summarize the recent advances in regeneration‐based therapeutic approaches using endogenous NSCs, including the results of our own collaborative groups.

Molecular pathogenesis of seipin/BSCL2-related motor neuron diseases

Heterozygous mutations in the Seipin/BSCL2 gene have recently been identified in two autosomal dominant motor neuron diseases, distal hereditary motor neuropathy type V and Silvers syndrome. Seipin protein is reportedly a transmembrane protein localized in the endoplasmic reticulum (ER). N88S and S90L mutations of this protein disrupt its glycosylation, resulting in its aggregation, but the mechanism of neurodegeneration remains unclear. To clarify the molecular pathogenesis of seipin‐related motor neuron diseases, we expressed wild‐type and mutant seipin proteins in neuronal and nonneuronal cells.

Characterization of seipin/BSCL2, a protein associated with spastic paraplegia 17.

Seipin, which is encoded by the BSCL2 gene, is a glycoprotein of unknown biochemical function that is associated with dominant hereditary motor neuron diseases. Mutations in the N-glycosylation site of seipin are associated with the disease states and result in accumulation of unfolded protein in the endoplasmic reticulum (ER), leading to the unfolded protein response (UPR) and cell death, suggesting that these diseases are tightly associated with ER stress. Here, we determined the subcellular localization, functional domains, and distribution of seipin in tissues. Our studies show that the transmembrane domains in seipin are critical for ER retention, ubiquitination, formation of inclusions, and activation of UPR. Using immunohistochemistry, seipin expression is detected in neurons in the spinal cord and in the frontal lobe cortex of the brain. The present study provides new insights into the biology of seipin protein that should help our understanding of the pathogenesis of seipin-related diseases.

Distribution and origin of TRPV1 receptor-containing nerve fibers in the dura mater of rat

We examined the distribution and origin of the nerve fibers innervating the dura mater of the rat that show immunoreactivity for the TRPV1 receptor (TRPV1-IR). Nearly 70% of the nerve fibers showing TRPV1-IR in the dura mater also exhibited CGRP-IR. Using a combination of immunohistochemistry and a retrograde tracer technique, we detected tracer accumulation in 0.6% of the neurons in the trigeminal ganglion and a few neurons in the dorsal root ganglion; half of the neurons in the trigeminal ganglion were small- and medium-sized (<or=1000 microm2). Among the tracer-accumulated neurons in the trigeminal ganglion, approximately 25% exhibited TRPV1-IR. Furthermore, nearly 80% of the tracer-accumulated small- and medium-sized neurons in the trigeminal ganglion that exhibited TRPV1-IR also exhibited CGRP-IR. Our findings indicate that the TRPV1 receptor in the dura mater and sensory ganglia may contribute to the pathophysiology of migraine, providing an important clue for the development of therapeutic strategies for migraine.

Oxidative metabolism in cultured rat astroglia: effects of reducing the glucose concentration in the culture medium and of D-aspartate or potassium stimulation

The glucose concentration in the culture medium may affect the energy metabolism of cultured cells. The oxidative metabolism of glucose in astrocytes might also be affected because the glucose concentration (25 mmol/L) of many culture media is higher than the physiological levels (∼3 mmol/L). In the present study, we assessed the effects of reducing the glucose concentration in the culture medium on the oxidative metabolism of glucose in cultured rat astroglia by measuring the oxidation rates of L-[U-14C]lactate or D-[U-14C]glucose to 14CO2. The effects of D-aspartate and elevated extracellular K+ levels on oxidative and glycolytic metabolism in astroglia were also investigated. The rates of [14C]lactate and [14C]glucose oxidation in astroglia cultured in a medium containing 2 mmol/L of glucose (astroglia2) were approximately twofold of those in astroglia cultured in a medium containing 22 mmol/L of glucose (astroglia22). D-Aspartate (500 μmol/L) significantly increased [14C]lactate oxidation by 156% in astroglia22 and by 83% in astroglia2. D-[U-14C]glucose oxidation in astroglia22 and astroglia2 was also increased by 94% and 76%, respectively. In contrast, an elevated extracellular K+ concentration (7.4 mmol/L) did not affect glucose and lactate oxidation, although it increased 2-deoxy-D-[1-14C]glucose phosphorylation. Astroglia grown in the physiological glucose concentration are more dependent on the oxidative metabolism of glucose than that in high-glucose concentration. Glucose concentration in culture medium has a strong influence on astrocytic oxidative capacity in vitro.

Initial oligemia with capillary flow stop followed by hyperemia during K+-induced cortical spreading depression in rats

Local cerebral blood volume (CBV) and capillary flow changes in regions of depolarizing neurons during K+-induced cortical spreading depression (CSD) in the cerebral cortex of α-chloralose-urethane-anesthetized rats were examined employing a transillumination (550u2009nm) video system. Capillary flow was calculated as the reciprocal of mean transit times of blood in pixels of 40u2009μm × 40u2009μm, each of which contains a few capillaries. Potassium microinjection into the cortex evoked repetitive wave-ring spreads of oligemia at a speed of ca. 2.33±0.48u2009mm/min. During the spread of CSD, tracer (either saline or carbon black) was injected into the internal carotid artery. Colocated with the oligemic wave, we detected capillary flow stop as evidenced by disappearance of the hemodilution curves. At any location in the region of interest within the cerebral cortex, we observed cyclic changes of capillary flow stop/hyperperfusion in synchrony with oligemia/hyperemia fluctuations. The initial flow stop and oligemia were ascribed to capillary compression by astroglial cell swelling, presumably at the pericapillary endfeet, since the oligemia occurred before larger vessel changes. We conclude that local depolarizing neurons can decrease adjacent capillary flow directly and immediately, most likely via astroglial cell swelling, and that the flow stop triggers upstream arteriolar dilatation for capillary hyperperfusion.

G501C polymorphism of oxidized LDL receptor gene (OLR1) and ischemic stroke

The human lectin-like oxidized low-density lipoprotein receptor 1 (OLR1/LOX-1) is the major endothelial scavenger receptor against oxidized low-density lipoprotein (Ox-LDL), which has been implicated in the pathogenesis of atherosclerosis. We investigated the G501C mutation in the OLR1 gene in 235 Japanese patients with ischemic cerebrovascular disease (CVD) and 274 age- and sex-matched healthy controls using single nucleotide primer extension analysis (SNuPe). There was no significant difference in the polymorphism between patients with ischemic CVD and controls (GC+CC versus GG, p=0.48). The C allele was not significantly different between the patients and controls (C versus G, p=0.91). Our results show that the OLR1 gene polymorphism has little effect on an increased risk for ischemic CVD in the Japanese population.