Leucine-rich α2-glycoprotein overexpression in the brain contributes to memory impairment.

We previously reported increase in leucine-rich α2-glycoprotein (LRG) concentration in cerebrospinal fluid is associated with cognitive decline in humans. To investigate relationship between LRG expression in the brain and memory impairment, we analyzed transgenic mice overexpressing LRG in the brain (LRG-Tg) focusing on hippocampus. Immunostaining and Western blotting revealed age-related increase in LRG expression in hippocampal neurons in 8-, 24-, and 48-week-old controls and LRG-Tg. Y-maze and Morris water maze tests indicated retained spatial memory in 8- and 24-week-old LRG-Tg, while deteriorated in 48-week-old LRG-Tg compared with age-matched controls. Field excitatory postsynaptic potentials declined with age in LRG-Tg compared with controls at 8, 24, and 48 weeks. Paired-pulse ratio decreased with age in LRG-Tg, while increased in controls. As a result, long-term potentiation was retained in 8- and 24-week-old LRG-Tg, whereas diminished in 48-week-old LRG-Tg compared with age-matched controls. Electron microscopy observations revealed fewer synaptic vesicles and junctions in LRG-Tg compared with age-matched controls, which became significant with age. Hippocampal LRG overexpression contributes to synaptic dysfunction, which leads to memory impairment with advance of age.

Leucine-rich α2-glycoprotein is a novel biomarker of neurodegenerative disease in human cerebrospinal fluid and causes neurodegeneration in mouse cerebral cortex.

Leucine-rich α2-glycoprotein (LRG) is a protein induced by inflammation. It contains a leucine-rich repeat (LRR) structure and easily binds with other molecules. However, the function of LRG in the brain during aging and neurodegenerative diseases has not been investigated. Here, we measured human LRG (hLRG) concentration in the cerebrospinal fluid (CSF) and observed hLRG expression in post-mortem human cerebral cortex. We then generated transgenic (Tg) mice that over-expressed mouse LRG (mLRG) in the brain to examine the effects of mLRG accumulation. Finally, we examined protein-protein interactions using a protein microarray method to screen proteins with a high affinity for hLRG. The CSF concentration of hLRG increases with age and is significantly higher in patients with Parkinson's disease with dementia (PDD) and progressive supranuclear palsy (PSP) than in healthy elderly people, idiopathic normal pressure hydrocephalus (iNPH) patients, and individuals with Alzheimer's disease (AD). Tg mice exhibited neuronal degeneration and neuronal decline. Accumulation of LRG in the brains of PDD and PSP patients is not a primary etiological factor, but it is thought to be one of the causes of neurodegeneration. It is anticipated that hLRG CSF levels will be a useful biomarker for the early diagnosis of PDD and PSP.

Chlamydophila pneumoniae in human immortal Jurkat cells and primary lymphocytes uncontrolled by interferon-γ.

Lymphocytes are a potential host cell for Chlamydophila pneumoniae, although why the bacteria must hide in lymphocytes remains unknown. Meanwhile, interferon (IFN)-γ is a crucial factor for eliminating chlamydiae from infected cells through indoleamine 2,3-dioxygenase (IDO) expression, resulting in depletion of tryptophan. We therefore assessed if lymphocytes could work as a shelter for the bacteria to escape IFN-γ. C. pneumoniae grew normally in human lymphoid Jurkat cells, even in the presence of IFN-γ or under stimulation with phorbol myristate acetate plus ionomycin. Although Jurkat cells expressed IFN-γ receptor CD119, their lack of IDO expression was confirmed by RT-PCR and western blotting. Also, C. pneumoniae survived in enriched human peripheral blood lymphocytes, even in the presence of IFN-γ. Furthermore, C. pneumoniae in spleen cells obtained from IFN-γ knockout mice with C57BL/6 background was maintained in a similar way to wild-type mice, supporting a minimal role of IFN-γ-related response for eliminating C. pneumoniae from lymphocytes. Thus, we concluded that IFN-γ did not remove C. pneumoniae from lymphocytes, possibly providing a shelter for C. pneumoniae to escape from the innate immune response, which has direct clinical significance.

Ciliates expel environmental Legionella-laden pellets to stockpile food.

When Tetrahymena ciliates are cultured with Legionella pneumophila, the ciliates expel bacteria packaged in free spherical pellets. Why the ciliates expel these pellets remains unclear. Hence, we determined the optimal conditions for pellet expulsion and assessed whether pellet expulsion contributes to the maintenance of growth and the survival of ciliates. When incubated with environmental L. pneumophila, the ciliates expelled the pellets maximally at 2 days after infection. Heat-killed bacteria failed to produce pellets from ciliates, and there was no obvious difference in pellet production among the ciliates or bacterial strains. Morphological studies assessing lipid accumulation showed that pellets contained tightly packed bacteria with rapid lipid accumulation and were composed of the layers of membranes; bacterial culturability in the pellets rapidly decreased, in contrast to what was seen in ciliate-free culture, although the bacteria maintained membrane integrity in the pellets. Furthermore, ciliates newly cultured with pellets were maintained and grew vigorously compared with those without pellets. In contrast, a human L. pneumophila isolate killed ciliates 7 days postinfection in a Dot/Icm-dependent manner, and pellets harboring this strain did not support ciliate growth. Also, pellets harboring the human isolate were resuscitated by coculturing with amoebae, depending on Dot/Icm expression. Thus, while ciliates expel pellet-packaged environmental L. pneumophila for stockpiling food, the pellets packaging the human isolate are harmful to ciliate survival, which may be of clinical significance.

A domino-like chlamydial attachment process: concurrent Parachlamydia acanthamoebae attachment to amoebae is required for several amoebal released molecules and serine protease activity.

Parachlamydia acanthamoebae is an obligate intracellular bacterium that infects free-living amoebae (Acanthamoeba), and is a potential human pathogen associated with hospital-acquired pneumonia. The attachment mechanism of this bacteria to host cells is crucial in bacterial pathogenesis, yet remains undetermined. Hence, we obtained monoclonal antibodies (mAbs) specific to either P. acanthamoebae or amoebae in an attempt to elucidate the attachment mechanism involved. Hybridomas of 954 clones were assessed, and we found that four mAbs (mAb38, mAb300, mAb311, mAb562) that were reactive to the amoebae significantly inhibited bacterial attachment. All mAbs recognized amoebal released molecules, and mAb311 also recognized the amoebal surface. mAbs reacted with the bacteria not only within amoebae, but also when they were released from amoebae (except mAb311). Furthermore, a serine protease inhibitor had an inhibitory effect on the bacterial attachment to amoebae, although none of the mAbs had any synergistic effect on the inhibition of attachment by the protease inhibitor. Taken together, we conclude that concurrent P. acanthoamebae attachment to amoebae is required for several amoebal released molecules and serine protease activity, implying the existence of a complicated host-parasite relationship.

Long-term oral lithium treatment attenuates motor disturbance in tauopathy model mice: implications of autophagy promotion.

Lithium, a drug used to treat bipolar disorders, has a variety of neuroprotective mechanisms including inhibition of glycogen synthase kinase-3 (GSK-3), a major tau kinase. Recently, it has been shown that, in various neurodegenerative proteinopathies, lithium could induce autophagy. To analyze how lithium is therapeutically beneficial in tauopathies, transgenic mice overexpressing human mutant tau (P301L) were treated with oral lithium chloride (LiCl) for 4 months starting at the age of 5 months. At first, we examined the effects of treatment on behavior (using a battery of behavioral tests), tau phosphorylation (by biochemical assays), and number of neurofibrillary tangles (NFTs) (by immunohistopathology). In comparison with control mice, LiCl-treated mice showed a significantly better score in the sensory motor tasks, as well as decreases in tau phosphorylation, soluble tau level, and number of NFTs. Next, we examined lithium effects on autophagy using an antibody against microtubule-associated protein 1 light chain 3 (LC3) as an autophagosome marker. The number of LC3-positive autophagosome-like puncta was increased in neurons of LiCl-treated mice. Neurons containing NFTs were completely LC3-negative, whereas LC3-positive autophagosome-like puncta contained phosphorylated-tau (p-tau). The protein level of p62 was decreased in LiCl-treated mice. These data suggested that oral long-term lithium treatment could attenuate p-tau-induced motor disturbance not only by inhibiting GSK-3 but also by enhancing autophagy in tauopathy model mice.

Expression analysis of high mobility group box-1 protein (HMGB-1) in the cerebral cortex, hippocampus, and cerebellum of the congenital hydrocephalus (H-Tx) rat.

High mobility group box-1 protein (HMGB-1), a protein expressed highly in developing neurons, is involved in the development and differentiation of neurons. At the same time, it functions as a transcriptional regulator of particular genes and as a cytokine: HMGB-1 released from a defective cell has been reported to induce damage to the adjacent cells.With a view to examine the relationship between neuronal damage caused by hydrocephalus and HMGB-1, we analyzed the expression of HMGB-1 in the cerebellum, cerebrum, and hippocampus of 1-day-old congenitally hydrocephalic H-Tx rats.As opposed to nonhydrocephalic H-Tx rats, the hydrocephalic H-Tx rats were observed to show stronger expression of HMGB-1 in the cerebellum, cerebrum, and hippocampus. Consequently, the protein was presumed to influence the development of neurons from an early postnatal stage not only in the cerebral cortex and hippocampus but also in the cerebellum, which is less susceptible to the direct effects of hydrocephalus. We expect that, in the future, regulating the expression or functions of HMGB-1 will lead to the possibility of impeding the progress of neuronal damage caused by hydrocephalus.