Misc

筋萎縮性側索硬化症(amyotrophic lateral sclerosis:ALS)は、大脳皮質運動野の上位運動ニューロンおよび脳幹と脊髄の下位運動ニューロンが選択的かつ系統的に障害される進行性の神経変性疾患である。ALSで障害されるα運動ニューロンは、構成する運動単位からFF(fast-twitch fatigable)、FR(fast-twitch fatigue-resistant)、S(slow-twitch)と3つのサブタイプに分類され、FF、FR、S型α運動ニューロンの順番で変性が生じる。近年、マトリクスメタロプロテアーゼ9(MMP9)がFF型α運動ニューロンに発現し、変性誘導に関与することが報告された。我々は、細胞外マトリクス(ECM)タンパク質であるオステオポンチン(OPN)が、MMP9とは異なるFR及びS型α運動ニューロンに発現することを発見し、OPNがALSの運動ニューロン変性に与える影響について検討した。ALSモデルマウス(SOD1G93Aマウス)脊髄中のOPNの局在を検討したところ、OPNは病態進行に伴い細胞外に放出され、ECMで粒子状構造物として観察された。またALS発症期の前後において、野生型マウスでは殆ど認められないOPN/MMP9共陽性の運動ニューロンが認められた。この共陽性の運動ニューロンは、FF型α運動ニューロンの変性(変性第1波)の後に代償的にリモデリングしたFR/S型α運動ニューロンであることを発見し、小胞体ストレスマーカーやOPN受容体であるαvβ3インテグリンの発現が認められたことから、ALS病態進行における運動ニューロン変性第2波の機序に、OPNによるインテグリンを介したMMP9活性化の関与が示唆された。またOPN欠損SOD1G93Aマウスは発症の遅延及び寿命の短縮を示し、OPNはALSの病態進行に対して2面性の性質を持つ可能性が示唆された。すなわち、OPNはALSの発症を規定する運動ニューロンに対しては障害的に、ALSの進行を規定するグリア細胞には保護的に作用する可能性が考えられた。以上より、我々はALSの第2波の運動ニューロン変性の機序としてOPN-インテグリン-MMP9系を新たに見出した。(著者抄録)

神経回路が修復されるメカニズムは、これまで、中枢神経系内の細胞および分子群の役割や、神経系細胞同士の連関を解明する形で、明らかになってきた。近年新たに、脈管系、免疫系、その他さまざまな臓器が産生する分子群が神経回路の修復に関与することが報告され、中枢神経系-末梢臓器間の連関という観点からさらなる研究が進められている。本稿では、疾患特異的な環境の変化である血管バリア機能の破綻に着目し、血管バリア機能の破綻に伴って流入する血液に含まれる、神経回路の修復作用をもつ分子について紹介する。(著者抄録)

The ability of choline to serve as a full, though low potency agonist for the α7 nicotinic receptor has provided the impetus to develop analogs that exhibit levels of potency and effectiveness suitable for use as therapeutic agents. Seven analogs of choline were synthesized based on previous work with the analog pyrrolidinecholine. The drugs were administered to differentiated PC-12 cells 24 hr prior to growth factor withdrawal which itself induced cytotoxicity in 30-40% of the cells. Three of 7 choline analogs exhibited potency and efficacy similar to that for nicotine as cytoprotective agents. Despite being tertiary amines, 4 of the choline analogs were more potent than choline in inhibiting [3H]choline uptake into cultured fibroblasts transfected with the high affinity, sodium-dependent choline transporter. One of the most effective analogs JAY 2-21-29 was shown to produce a potent (EC50 ∼ 30 nM) cytoprotective action that was blocked by pretreatment with the α7 nicotinic receptor selective antagonist methyllycaconitine, but not by theα2 subtype-preferring antagonist dihydro-β-erythroidin. These preliminary studies support the further neurochemical characterization of these compounds 1) as selectiveα7 nicotinic receptor agonists and, 2) based on their interaction with the choline transporter, as potential cholinergic false neurotransmitters as has been demonstrated for pyrrolidinecholine.

Seven types of mRNA that differed in the 5′-non-coding region were identified for choline acetyltransferase of mouse spinal cord. These mRNAs were produced by differential splicing of pre-mRNAs transcribed from three different promotor regions. Two murine cholinergic cell lines, NS20Y and NG108-15, expressed M-type mRNA most abundantly. Using these cell lines, promotor activity in choline acetyltransferase gene was analyzed by transient assay of a reporter gene. The result indicated that there was promotor activity in the region upstream of the M-type exon and enhancer activity in the intron downstream of the M-type exon, and that this region regulated neuron-specific expression of choline acetyltransferase activity. In contrast, R-type mRNA was exclusively expressed in cultured superior cervical ganglion cells and was markedly increased by ciliary neurotrophic factor (CNTF). To analyse the physiological role of CNTF, we constructed and screened a cDNA library from human sciatic nerves and isolated two types of cDNAs for human CNTF. Sequence analysis revealed that one type of cDNA corresponded to the normal mRNA, while the other type contained a 4 bp stretch insertion within the coding region, which caused frameshift from 39th amino acid with a stop codon 24 amino acids downstream. Analysis of genomic DNA for CNTF showed that there was a point mutation from G to A in the intron of the mutated allele, which created a new splice acceptor site and generated a new mRNA species with 4 bp insertion. Analysis of tissue samples from autopsied subjects and transfection study of CNTF minigenes into cultured cells indicated that the mutated allele expressed only the mutated mRNA, but not the normal mRNA species. Thus, this allele represents a null mutation that abolishes expression of normal CNTF protein. CNTF genotypes were analyzed in 400 Japanese subjects. The frequency of three genotypes did not differ between healthy volunteers and patients suffering from various neurological diseases. Nine mutant homozygote subjects so far detected did not show any neurological disturbances and developmental abnormalities until adulthood. These observations indicate that CNTF is dispensable for the development and maintenance of the nervous system, and that the CNTF null mutation is not causally related with any neurological disease.