Kiminobu Sugaya

Nitric oxide synthase gene expression in cholinergic neurons in the rat brain examined by combined immunocytochemistry and in situ hybridization histochemistry.

The expression of mRNA for the calmodulin-dependent form of brain nitric oxide synthase (NOS) was examined in cholinergic cells of the rat brain using a method combining in situ hybridization histochemistry with immunocytochemistry for choline acetyltransferase (ChAT) in the same brain sections. We constructed a riboprobe specific for brain NOS by subcloning a 493 bp fragment of the coding region which displayed low homology to other forms of NOS. The general distribution of NOS mRNA was in excellent agreement with previous studies using the full-length probe or NADPH diaphorase histochemistry. NOS mRNA was observed in many brain structures and relative levels were quantitated using grain counting procedures in a number of cholinergic and non-cholinergic neuronal groups throughout the brain. In the forebrain, ChAT-immunoreactive cells or cell groups were observed in medial septum (MS), vertical limbs of diagonal band (DBV) and horizontal limbs of diagonal band (DBH), nucleus basalis magnocellularis (NBM), substantia innominata (SI), and striatum (ST). In the brainstem, the cholinergic groups studied included those located in the pedunculopontine tegmental nucleus (PPTN), the laterodorsal tegmental nucleus (LDTN), the nucleus parabigeminalis and several motor nuclei. For NOS mRNA quantitation, silver grains overlying ChAT-stained neuronal profiles in sections on emulsion-dipped slides were counted digitally. In the LDTN and PPTN, virtually all the ChAT-positive cells expressed NOS mRNA at high levels. In MS, DBV and SI, about 30-50% of the ChAT-positive cells expressed NOS mRNA at low-to-moderate levels. Less than 20% of ChAT-positive neurons in the other cholinergic populations studied expressed NOS mRNA; the NBM was one of these low-expressing populations. Many scattered non-cholinergic cells expressing NOS mRNA were found in the striatum and cerebral cortex. In other non-cholinergic regions, high NOS mRNA expression was observed in the islands of Calleja, thalamic and hypothalamic nuclei, several amygdaloid nuclei, regions related to the optic tract, the interpeduncular nucleus, and the supramammillary nucleus. The heterogeneous distribution of NOS mRNA implies complex roles for nitric oxide neurotransmission in brain function, including for the cholinergic phenotype. Additionally, given the postulated involvement of nitric oxide in neurodegeneration, the widely varying levels of expression of NOS within identified central cholinergic neurons may relate to differential vulnerability of this phenotype in disease or aging.

Human neural stem cells improve cognitive function of aged brain.

The capability for in vitro expansion of human neural stem cells (HNSCs) provides a well characterized and unlimited source alternative to using primary fetal tissue for neuronal replacement therapies. The HNSCs, injected into the lateral ventricle of 24-month-old rats after in vitro expansion, displayed extensive and positional incorporation into the aged host brain with improvement of cognitive score assessed by the Morris water maze after 4 weeks of the transplantation. Our results demonstrate that the aged brain is capable of providing the necessary environment for HNSCs to retain their pluripotent status and suggest the potential for neuroreplacement therapies in age-associated neurodegenerative disease.

Septo-hippocampal cholinergic and neurotrophin markers in age-induced cognitive decline

Messenger RNA (mRNA) molecules encoding proteins related to the presynaptic cholinergic and neurotrophin systems were quantitated in the hippocampus and basal forebrain of Long-Evans rats with spatial learning ability assessed in the Morris water maze. The reverse transcriptase-polymerase chain reaction showed that the mRNAs for the low-affinity neurotrophin receptor (p75-NTR) and the growth-associated protein GAP-43 were decreased in level in the basal forebrain of aged-impaired rats. In the hippocampus of these aged-impaired rats, the mRNA for VGF, another neurotrophin-inducible gene, also was decreased. In situ hybridization histochemistry revealed that mRNAs for nerve growth factor (NGF) and brain-derived neurotrophic factor increased in level in the aged rat hippocampus; when age effects were removed, NGF mRNA level remained significantly correlated with maze performance. Enzyme-linked immunosorbent assay indicated that NGF protein was expressed at normal levels in the aged rat hippocampus. These mRNA and protein alterations may signify that a defect in neurotrophin signaling exists in the brains of aged Long-Evans rats, underlying reduced plasticity responses in the basal forebrain cholinergic system.

Survival and plasticity of basal forebrain cholinergic systems in mice transgenic for presenilin-1 and amyloid precursor protein mutant genes.

The basalo-cortical cholinergic system was characterized in mice expressing mutant human genes for presenilin-1 (PS1), amyloid precursor protein (APP), and combined PS/APP. Dual immunocytochemistry for ChAT and Aβ revealed swollen cholinergic processes within cortical plaques in both APP and PS/APP brains by 12 months, suggesting aberrant sprouting or redistribution of cholinergic processes in response to amyloid deposition. At 8 months, cortical and subcortical ChAT activity was normal (PS/APP) or elevated (PS, APP frontal cortex), while cholinergic cell counts (nBM/SI) and receptor binding were unchanged. ChAT mRNA was up-regulated in the nBM/SI of all three transgenic lines at 8 months. The data indicate that the basal forebrain cholinergic system does not degenerate in mice expressing AD-related transgenes, even in mice with extreme amyloid load. The PS1 or APP transgene appears to enhance the cholinergic phenotype in younger mice, but this involves aberrant sprouting and redistribution of cortical cholinergic processes.

In vitro differentiation of multipotent human neural progenitors in serum-free medium.

Stem cells are exciting candidates for therapeutic strategies in neurodegenerative diseases, due to their multipotency and migratory capabilities. We show that stem cell-like embryonic normal human neural progenitors (HNPs) are capable of proliferating in response to mitogenic growth factors and differentiate into diverse CNS cell types in vitro. We present evidence that HNPs differentiate to βIII-tubulin-, glial fibrillary acidic protein- and O4- immuno-positive cells, in both a fetal bovine serum-containing and a non-supplemented, serum-free basal medium. From these findings, we propose that HNPs may differentiate not only in response to exogenous differentiation factor(s) contained in the serum, but also in response to some endogenous factor(s) released from the HNPs, which may regulate the differentiation pathway of these cells.

Indicators of glial activation and brain oxidative stress after intraventricular infusion of endotoxin.

Glial activation and oxidative stress are both consequences of brain aging. To investigate whether glial activation causes oxidative stress or not, the immune activator, lipopolysaccharide (LPS), was intraventricularly injected into the rat brain. The expression of candidate genes were examined by in situ hybridization histochemistry (ISHH) combined with immunohistochemistry for glial markers over a period of time up to 24 h after the LPS injection. The mRNA for glial fibrillary acidic protein (GFAP) was elevated around the injection site by 2 h, and the volume of elevated expression spread to the entire brain after 6 h, with higher levels present in the injected hemisphere. The level of inducible isoform of nitric oxide synthase (i-NOS) mRNA increased in a punctate-like pattern in the region of the injection by 6 h and this response spread to the entire brain after 12 h. These results indicate that the glia are activated for at least 24 h after a single LPS injection. The mRNAs for a heat-shock protein (HSP70) and for the manganese-dependent superoxide dismutase (Mn-SOD) were elevated in the ipsilateral hemisphere as early as 2 h post-injection, but these responses subsided nearly to basal levels by 4 h. These levels of mRNAs for these genes increased again after 6 h of the LPS injection; thus, the earlier increases of the messages appeared to be associated with the survival surgery procedure. With microautoradiographic analysis, scattered OX-42 positive cells expressed i-NOS mRNA after 6 h post-injection, but elevation of Mn-SOD mRNA was not detected in either microglia or astrocytes at any time point examined. The level for Cu/Zn-SOD mRNA did not alter at any time point. The beta-amyloid precursor protein (betaAPP) mRNAs were elevated beginning at 6 h. These results indicate that chronic glial activation leads to a condition of oxidative stress in the brain. The data also suggest that LPS injection could be used to study the effects of chronic glial activation on the survival of neuronal populations that could be at risk from oxidative stress.

Topographic associations between DNA fragmentation and Alzheimer's disease neuropathology in the hippocampus

To identify whether the process of apoptosis bears a topographic relationship to selected aspects of Alzheimers disease (AD) pathology, we used an in situ nick translation method (TUNEL) to map DNA fragmentation in hippocampal sections immunostained for abnormally phosphorylated tau, which exists in the neurofibrillary tangles (NFTs) and in the dystrophic neurites associated with senile plaques. To ascertain associations of DNA fragmentation with glia, TUNEL was combined with immunohistochemistry for the astrocyte marker, glial fibrillary acidic protein (GFAP), or the microglial antigen OX-42. Consistent with previous reports, the incidence of putative DNA fragmentation detected by TUNEL was much higher in the AD brain, compared to non-demented subjects. While most TUNEL-positive cells did not exhibit any systematic topographic relationship to senile plaques, which were visualized by immunostain of abnormally phosphorylated tau for dystrophic neurites, DNA fragmentation was found frequently within cells containing NFTs. In hippocampal sections prepared to visualize glia, DNA fragmentation was not observed in GFAP-positive astrocytes, but some OX-42-positive microglia exhibited TUNEL signals. Other TUNEL-positive cells were found frequently in proximity to glia. The data suggest that cells compromised by the deposition of NFTs are prone to initiate the process of apoptosis. Furthermore, some glial populations appear to be apoptotic in the AD brain.

Differential vulnerability of primary cultured cholinergic neurons to nitric oxide excess

Many neuronal nitric oxide synthase (nNOS)-expressing brain neurons, including some cholinergic populations, are resistant to disease or to certain forms of excitotoxicity. Vulnerability to NO excess of forebrain (medial septal/diagonal band; MSACh) and brainstem (pedunculopontine/laterodorsal tegmental nuclei; BS-ACh) cholinergic neurons was compared in E16-E18 primary rat brain cultures. MS-ACh cells were ∼300-fold more sensitive to the NO donor S-nitro-N-acetyl-D,L-penicillamine (SNAP) than were BS-ACh cells. Most (69%) MS-ACh cells contained nuclear DNA fragments by 2 h after addition of SNAP, while only 21% BS-ACh cells were TUNEL-positive after NO excess. Depletion of glutathione content did not potentiate the effect of SNAP on MS-ACh cells, but sensitized BS-ACh cells to the NO donor. Caffeic acid, a putative NF-KB inhibitor, enhanced the toxicity of SNAP to cholinergic neurons in both preparations. Our experiments show that cholinergic neurons in mixed primary cultures from different brain regions possess biochemical differences with respect to their vulnerability to NO excess.

mRNA for the m4 muscarinic receptor subtype is expressed in adult rat brain cholinergic neurons

A number of pharmacological, anatomical, and immunological studies have previously addressed the subtype identity of the hippocampal muscarinic pre-synaptic autoreceptor. A preponderance of findings indicate that it is of the M2 pharmacological type. Both the m2 and m4 molecular subtypes exhibit M2 pharmacology and there are few drugs that differentiate between these receptors. Pharmacological attempts at defining the hippocampal autoreceptor have yielded conflicting results. The basal forebrain is relatively enriched in m2 muscarinic receptor mRNA and protein, and lesions that denervate the hippocampus of its basal forebrain cholinergic input have shown a decrement in m2, but not m4, receptor protein in the hippocampus. Thus, the anatomical data obtained to date tend to support the view that the m2 subtype is expressed as the hippocampal autoreceptor. We have combined in situ hybridization histochemistry (ISHH) with immunocytochemistry to choline acetyltransferase to examine whether mRNA for the m4 subtype of muscarinic receptor is expressed in central cholinergic neurons. The m4 muscarinic mRNA was found at moderate levels in all subdivisions of the cholinergic basal forebrain, including the medial septum/diagonal band complex (MS/DB). The m4 mRNA was also found in striatal cholinergic interneurons, in the cholinergic reticular core of the upper brainstem, and in brainstem cholinergic motor neurons. Muscarinic m4 receptor mRNA was also found in many non-cholinergic cells in the brain. For example, the hippocampal pyramidal neurons, dentate gyrus granule cells, and entorhinal cortical pyramidal neurons express relatively high levels of m4 mRNA, while in the brainstem the dorsal raphe and pontine reticular nuclei express relatively high levels of this mRNA. The finding of m4 mRNA in the MS/DB cholinergic neurons suggests that this receptor protein might be expressed as an autoreceptor in hippocampal cholinergic terminals.

Induction of manganese superoxide dismutase in BV-2 microglial cells

THE regulation of the manganese-dependent superoxide dismutase (Mn-SOD) was studied in immortalized microglial cells (line BV-2). BV-2 cells, activated with lipopolysaccharide (LPS), exhibited an increase in MnSOD-like immunoreactivity, that was associated with an accumulation of nitrite in the culture medium and an increase in immunoreactivity for the inducible type of nitric oxide synthase (i-NOS). The i-NOS inhibitor L N6-(1-iminoethyl)-lysine (NIL, 600 μM) suppressed the nitrite accumulation and the increase in Mn-SOD-like immunoreactivity in activated cells without significant effect on the level of i-NOS-like immunoreactivity. The NO donor sodium nitroprusside dose-dependently increased Mn-SOD-like immunoreactivity in NILpretreated BV-2 cells. These results indicate that the induction of Mn-SOD in activated BV-2 cells is mediated in part by NO, or its metabolites.