Beta caryophyllene (BCP) is one of the sesquiterpenes found abundantly in cannabis as well as other plants such as clove, rosemary, black pepper, and lavender. Past studies have shown this terpene has antioxidant and anti-inflammatory effects, which is believed to be mediated by cannabinoid receptor type 2 (CB2). Recent study showed the attenuation of aortic inflammation by BCP inhalation, which is abolished by the treatment of CB2 antagonist.

CB2 receptor is the Gi/Go type GPCR majorly expressed on the immune-related cells throughout the body. Thus, CB2 has been reported to act as a regulator in the immune system, but not many researches has been conducted to see the therapeutic possibility of CB2 to treat e.g. autoimmune disease by their immune suppression properties. However, since BCP showed inflammatory suppression effect by inhalation, we expected that BCP might be able to attenuate other inflammatory responses, such as allergic diseases through CB2-mediated immune suppression. We therefore examined whether CB2 can suppress allergic responses, particularly by acute inhalation of BCP. The BCP inhalation attenuated sneezing and nose scratch in nasal allergy model mice. Further, the immune cell population of CB2-deficient mice shifted similarly to that of wild-type mice after BCP administration. Altogether, we propose that BCP could be a potential substance to cure allergic diseases.

It is widely known that cannabinoid type 2 (CB2) receptor deficiency enhances inflammatory response and further symptoms in various animal models of inflammation, allergy, or cancer. As CB2 receptors are inhibitory Gi/Go G-protein coupled receptors and as major expression site of CB2 receptors are immune cells, it is no wonder that lack of CB2 receptor might lead the exacerbated inflammation. We therefore hypothesized that lack of CB2 receptor might also enhance the high fat diet (HFD)-induced peripheral neuroinflammation. However, surprisingly, CB2 receptor knockout animals (CB2-KOs) showed the significant resistance to the HFD-induced neuroinflammation. Namely, 5-week feeding of HFD induced substantial hypersensitivity in WT mice, while tactile sensitivity of HFD-fed CB2-KO remained intact. In the same animals, we further found the robust upregulation of infiltrated macrophages, chemokine receptor CXCR4 expression and modified differentiation of splenic myeloid-derived suppressor cells (MDSCs) in HFD-fed WT animals, but not in either HFD-fed CB2 knockout mice or standard fat diet (SFD)-fed WT and CB2-KO controls. Based on these results, we will propose that CB2 receptors might have the bipolar regulatory role to chemokine receptor-mediated inflammatory response through the modulation of splenic MDSC differentiation, which in the end enhance or inhibit the development of neuroinflammation depending on its cause.

Cannabinoid CB1 receptor is known to play an important role on the neuronal development such as columnar formation in cortex or age-related changes of cognitive function. Interestingly, aged CB1 receptor-deficient mice show a marked cognitive decline, whereas young CB1 knockouts show better cognition than wild-type mice. However how CB1 influence the neuronal function in very young age like infants is unknown, majorly due to lack of in-vivo/ex-vivo evaluation systems such as behavioral tests and primary cell cultures in this particular age. Thus, this study aims to generate primary cultured neurons from infant-to-toddler mice (2-3 weeks old), which has been difficult to do so far, and to evaluate whether they can be used as an ex-vivo model to analyze the neural activity these mice.

We used modified version of manufacturer’s protocol by Miltenyi Biotec. While this protocol is designed to culture the neurons from adult mice up to p60, our modified protocol could culture the neurons from neonatal (P7), young (P50), and mature (P105) mice at least for 3 to 10 days which was good enough to grow axons. This result suggests that our modified method is suitable to evaluate neuronal growth and cell activity even after a certain ageing period like P105. However, since our method is restricted to the whole brain, we are currently trying to culture neurons harvested from specific brain regions. Our final goal is to establish the primary cultures from infant CB1 knockouts as well as DAGL-alpha knockouts, to study the effects of CB1-related endocannabinoid deficiency on neuronal growth and cell activity in infants-toddlers.

諸外国における大麻の条件付き解禁の流れを受けて、大麻の生理活性物質であるカンナビノイドおよび内因性カンナビノイド系が注目されている。中でもカンナビジオール（CBD）は向精神作用を持たず、かつ乳幼児のてんかん発作のような既知の薬物だけでは対応しきれない症例にも奏効したことから、同様に既知の医薬品では治療できない他の疾患や不定愁訴に対し効果があるのではないか、という点において大きな注目を浴びている。医薬品として（他国において）承認されているCBD製剤としてはエピディオレックスがあるが、それ以外にも「大麻由来の植物成分」という触れ込みのキャッチーさから、セルフケアを目的とした様々な製品が生産・販売されており、2023年度の世界市場はゆうに1兆円を超える巨大市場と化している。しかし市場が指数関数的に巨大化していく一方で、臨床研究および基礎研究は未だ十分であるとはいえず、十分なエビデンスがあるといえるのは先述したエピディオレックスの適応症となっている難治性てんかんおよび結節性硬化症くらいである。このため今も世界中で様々な臨床および基礎研究が盛んに進められており、その中で新たな適応症候補も数多く上がっている一方、思わぬ副作用も様々に報告されて来ている。本項ではそういった「基礎研究から新たに浮かび上がってきた様々な適応症候補および思わぬ副作用」を紹介すると同時に、「CBDという成分」を基礎研究や臨床応用に使う際に考慮すべき点も併せて論じたい。

There are 40 million people worldwide living with Alzheimer's Disease (AD), and by 2050 the prevalence is expected to increase to 150 million people, thus resulting in a 1 in 2 chance of having AD by the age of 85. An increase in amyloid-b plaques (Ab) and hyperphosphorylated tau are widely accepted as the core indicators of the disease. These indicators of AD onset and progression are becoming increasingly attributed to the disturbance of optimal sleep/wake cycles. Additionally, AD progression exacerbates normal sleep/wake cycles, resulting in a cyclical worsening of AD pathology, circadian rhythm, and cognitive dysfunction. By using an Ab intracerebroventricular (ICV) injection mouse model, mice can present an AD-like pathology within days to weeks after injection, allowing for the expedited examination of the disease. This model can serve as a tool to investigate the mechanism behind the disturbance of the biological clock and AD progression, and therefore find therapeutic targets to delay, prevent or cure the disease.

Cannabinoid receptor type 2 (CB2) is one of the major receptors for cannabis, which is expressed all over the body especially on the immune-related cells. It is considered that CB2 can work as a regulator in the immune system, particularly to suppress inflammatory responses in macrophages and microglias when activated. However no other immune cells have been studied whether and how they get modulated by CB2 activity. The objective of this experiment is to search for the other immune cell that can be affected by CB2 activity, and reveal the detailed mechanism of CB2-involved immune regulation.

To characterize the CB2-regulated cells under the inflammatory state, we injected LPS (1 mg/kg, i.p.) to CB2 deficient mice (CB2-KO) and wild-type (WT) controls. Spleen has been harvested from these mice 2 hours after the LPS administration, and immunophenotyping by flow cytometry has been conducted. In this experiment, we found the particular increase of the type 2 classical dendritic cell (cDC2) population in LPS-treated CB2-KO. As past studies reported that cDC2 recruits helper T cells when the body is exposed to an allergen, we further induced allergic rhinitis by OVA sensitization to investigate in-vivo effect of cDC2 increase in CB2-KO and WT animals. As expected, CB2 deficiency resulted as the significant exacerbation of allergic symptoms compared to WT mice. These results suggest that CB2 activity may suppress the allergic response by the reduction of cDC2 recruitment. Pharmacological effect of CB2 agonists to these symptoms will be studied as the future experiment.

カンナビノイドが、今、熱い。その「大麻（カンナビス）」という言葉の響きもあってか、あるいは大麻が持つという様々な難治性疾患への治療効果を期待するからか、現在世界中で大麻由来成分＝カンナビノイドを使った様々な製品が爆発的に流行している。現在のところ、他の医薬品などと比べて「確実にカンナビノイドの方が優れている」という科学的根拠はほぼ無いにも関わらず、である。とはいえ確かにある種の疾病の病態を改善し、QOLを上げるということは、例えば患者自身の経験として世に語られていたりする。彼らは一様に「エンドカンナビノイドシステムが鍵だ」というが、さて、一方でエンドカンナビノイドシステムがどこまで理解されているかは、また別の話であろう。エンドカンナビノイドシステム、日本語では一般的に内因性カンナビノイド系と呼ばれて来たシグナル伝達系は、大麻の活性成分が特異的に結合する2つのGタンパク質（Gi/o）共役型受容体CB1とCB2受容体と、これらの受容体の各々に対応する内因性リガンド、そしてこれらリガンド軍の生成酵素や分解酵素などから構築されている。長年CB1は脳や中枢神経系に、CB2は末梢の免疫系細胞に発現していると考えられて来たが、実際のところどちらの受容体も、ある程度の偏りはあるものの、全身に極めて広範に分布しており、それぞれの発現箇所で代謝系・神経系・免疫系など実に様々な生理機能を調節していることが、最近の研究で解って来た。さてここで1つお伺いしたい。これだけあちこちに広範に発現し、様々な生理機能の制御・調節を担うカンナビノイド受容体だが、ではこれらの受容体が無くなってしまったら、我々の身体は一体どうなってしまうのだろうか。つまり内因性カンナビノイド系が無いと、我々の身体には何がおきるのだろうか。我々はこの普遍的な問いに答えるべく、受容体を欠損させた遺伝子改変動物を用いて様々な検討を行ってきた。本講演ではその中でもCB1受容体の欠損が「加齢」や「肥満」に及ぼす影響、またCB2受容体の欠損が「感染症」や「慢性疼痛」にどのような影響を及ぼすかを、我々の最近の知見も併せて紹介する。

It is indicated that the intestinal environment affects the brain, which is known as the gut-brain axis. In fact, improvement of intestinal environment is reported to suppress anxiety-like behavior in mice model of depression and schizophrenia. However, its detailed mechanisms remain unclear, particularly in context of intestinal environment effects on emotion in non-disease mice. The object of this experiment is therefore to verify the effect of intestinal environment on anxiety-like behavior and its detailed physiological mechanisms.

We previously found that cellulose rich food (AIN-93M) suppresses the production of short-chain fatty acid and exacerbate the intestinal condition. In the present study, we fed mice with AIN-93M to modify the intestinal environment, or the mouse chow that can maintain a favorable intestinal environment (MF). After 8-weeks feeding, AIN-93M-fed animals displayed the significant increase of marble-burying behavior compared to MF-fed group, suggesting that dysfunction of intestine lead by AIN-93M enhanced anxiety-like behavior. Furthermore, dopamine release as well as dopamine receptor expression has been increased in amygdala of AIN-93M fed mice but not in MF animals, indicating that enhanced anxiety-like behavior in AIN-93M animals is due to such modification of amygdalar dopaminergic system. 

These results suggest that cellulose rich food may exacerbate intestinal environment, which may enhance the anxiety-like behavior and overactivation of dopaminergic system.

It is widely known that cannabinoid type 2 (CB2) receptor deficiency enhances neuroinflammation and pain development in the animal model of nerve injury-evoked neuropathic pain. We previously proposed the upregulated leptin signaling at the peripheral nerve as one of the underlying molecular mechanism, as nerve-injured CB2 receptor knockouts (CB2-KO) displayed robust upregulation of leptin receptors in both injured and non-injured nerve tissue. Those leptin receptors seemed to be expressed on the macrophages which is recruited to the nerve by the tissue injury, indicating the infiltration of leptin receptor-expressing macrophages. Thus, Due to these past results we also hypothesized that lack of CB2 receptor might also enhance the high fat diet (HFD)-induced peripheral neuroinflammation. However, surprisingly, CB2-KOs showed the significant resistance to the HFD-induced neuroinflammation. Namely, 5-week feeding of HFD induced substantial hypersensitivity in WT mice, while tactile sensitivity of HFD-fed CB2-KO remained intact. In the same animals, we further found the significant upregulation of infiltrated macrophages and chemokine receptor CXCR4 expression in HFD-fed WT animals, but not in either HFD-fed CB2 knockout mice or standard fat diet (SFD)-fed WT and CB2-KO controls. Based on these results, we will propose that CB2 receptors might have the bipolar regulatory role to chemokine receptor-mediated inflammatory response, which in the end enhance or inhibit the development of neuroinflammation depending on its cause.

Recent study showed that cannabinoid CB1 receptor-mediated signaling may take a huge role in age-related modification of brain function. CB1 deficient animals show significant cognitive deficit in old age, however they show better learning ability in young age. Even though the finding is surprising, the molecular mechanisms of such difference, especially for better learning in young age remains unknown mainly due to lack of suitable in-vivo/ex-vivo models. Here we will purpose to use the primary cultured neurons as the ex-vivo model of aging, which can be enabled with gentleMACS technology. This method is said to enable the primary cell culture from adult rodents but up to age of P60. In the present study we aimed to establish a method for primary culture of brain neurons using aged mice, which can be also used for knockout animals of CB1 receptors.

We first tried to prepare the primary culture of brain neurons from neonatal (P7), young (P50) and mature (P105) mice. After 3-days of culture we could find that all of them survived well and start to extend their axons. Notably, P105 is twice an older age than previously reported suggesting that this method could be usable to evaluate the neuronal growth and cellular activity after the certain aging. Further, they could even survive for next 10-days with continuous axon growth. Using this method, we will further conduct the primary cell culture in knockout animals of CB1 as well as endocannabinoid producing enzyme DAGL-a to see the effect of endocannabinoid deficiency to the neuronal growth and cellular activity.

Cannabidiol (CBD) is the second major cannabinoid which is often said to improveanxiety and sleep with no psychoactive effect. It is said that CBD can promotethe production of wake-related neurotransmitters such as dopamine. Further,co-injection of CBD and THC increased sleeping time in rats. However, no studyhas been done for the in-vivo effect of CBD to circadian clock. Present studyaimed to elucidate whether CBD can modify peripheral circadian rhythm.

PER2::LUC knock-in female mice were used to determine the effect of CBD to theperipheral clock. Mice were divided into six groups: CBD isolate (99%crystalline) in MCT oil, CBD isolate in 5% EtOH/5% cremophor/water, andwater-soluble CBD nanopowder in water, with respective vehicle controls. Eachdrug was orally administered at ZT4 or ZT16 for three days, then PER2 geneexpression in the liver, kidney, and submandibular gland is observed by in-vivoimaging.

We found that phase advance in the liver and the submandibular gland only happenswhen MCT or CBD in MCT (CBD/MCT) was administered at ZT4. However, the phaseadvance did not differ between the MCT and CBD/MCT groups. Furthermore, neitherthe vehicle nor the CBD affected the peripheral clock when administered atZT16. 

This study suggested that not CBD but rather MCT oil affect the circadian clockin mice. As MCT oil is commonly used as a base for CBD products, we propose thatMCT might be the possible factor that affects the circadian clock and causerhythm improvement. Therefore further detailed studies on the effect of CBDproducts will be needed.

博士号取得後フランスを経てドイツに流れつき、運良く研究費を得てチームリーダーにまでなった私の次の挑戦は「お母さんになること」だった。ドイツならではの医療制度および研究助成制度に助けられ倒して妊娠・出産・職場復帰までこぎつけた経緯を、そもそもなぜ欧州に飛ぶことになったのかも含めて紹介する。ご笑覧いただきたい。