L-type calcium channel blockers and substance P induce angiogenesis of cortical vessels associated with beta-amyloid plaques in an Alzheimer mouse model

AbstractIt is well established that L-type calcium channels (LTCCs) are expressed in astroglia. However, their functional role is still speculative, especially under pathologic conditions. We recently showed that the α1 subunit-like immunoreactivity of the CaV1.2 channel is strongly expressed in reactive astrocytes around beta-amyloid plaques in 11-month-old Alzheimer transgenic (tg) mice with the amyloid precursor protein London and Swedish mutations. The aim of the present study was to examine the cellular expression of all LTCC subunits around beta-amyloid plaques by in situ hybridization using 35S-labeled oligonucleotides. Our data show that messenger RNAs (mRNAs) of the LTCC CaV1.2 α1 subunit as well as all auxiliary β and α2δ subunits, except α2δ-4, were expressed in the hippocampus of age-matched wild-type mice. It was unexpected to see, that cells directly located in the plaque core in the cortex expressed mRNAs for CaV1.2 α1, β2, β4, and α2δ-1, whereas no expression was detected in the halo. Furthermore, cells in the plaque core also expressed preprotachykinin-A mRNA, the precursor for substance P. By means of confocal microscopy, we demonstrated that collagen-IV-stained brain vessels in the cortex were associated with the plaque core and were immunoreactive for substance P. In cortical organotypic brain slices of adult Alzheimer mice, we could demonstrate that LTCC blockers increased angiogenesis, which was further potentiated by substance P. In conclusion, our data show that brain vessels associated with beta-amyloid plaques express substance P and an LTCC and may play a role in angiogenesis

Reactive astrocytes surrounding -amyloid [beta-amyloid ] plaques in a mouse model of Alzheimers disease : emphasis on L-type calcium channels, neuronal survival and angiogenesis

Alzheimer-Demenz (AD) zählt zu den häufigsten neurodegenerativen Erkrankungen bei Menschen mit fortgeschrittenem Alter. Diese Krankheit ist charakterisiert durch verschiedene physiologische Veränderungen im Gehirn wie (1) die Anhäufung von extrazellulären -amyloid (A) Plaques, (2) die intrazelluläre Bildung von hyperphosphorylierten tau tangles, (3) den Verlust von Synapsen und daraus resultierendem Zelltod cholinerger Neurone und (4) die Aktivierung von Astrozyten sowie Mikroglia, welche zu entzündlichen Prozessen führen kann. Es ist mittlerweile bekannt, dass die AD auch zu einer Dysbalance im Kalziumhaushalt beitragen kann. Des weiteren wurde anhand eines Mausmodells für AD herausgefunden, dass A Plaques von reaktiven Astrozyten umgeben sind, welche eventuell an der gestörten Kalzium-Homöostase im Gehirn beteiligt sind. Das Ziel der vorliegenden Dissertation war es (1) die Expression von spannungsabhängigen L-typ-Kalziumkanal (engl. L-type calcium channel, LTCC) Untereinheiten in reaktiven GFAP (saures Gliafaserprotein) positiven, A Plaques umgebenden Astrozyten mit Hilfe eines AD Mausmodells zu untersuchen, (2) die mRNA Expressionsprofile von LTCC Untereinheiten in primären Mausastrozyten zu analysieren und (3) die Auswirkungen von Kalziumkanalblockern (engl. Calcium channel blockers, CCBs) auf axotomierte cholinerge und dopaminerge Neurone in organotypischen Rattenhirnschnitten zu studieren. Anhand dieser Dissertation konnte aufgezeigt werden, dass die Hochregulierung der LTCC CaV1.2 1 Untereinheit in reaktiven Astrozyten mit dem Auftreten von A Plaques in transgenen 11 Monate alten AD Mäusen assoziiert ist. Zudem wurde festgestellt, dass die Auxiliaruntereinheit 4 nicht mit reaktiven Astrozyten kolokalisierte. Mittels in-situ Hybridisierung konnte gezeigt werden, dass die mRNA für CaV1.2 1, 2, 4 und 2-1 hauptsächlich im Kernbereich des A Plaques exprimiert wird, was wiederum einen funktionellen LTCC darstellen könnte. Die mRNA, welche für das Neuropeptid Preprotachykinin A kodiert, ein Vorläufermolekül für Substanz P, konnte ebenso im Inneren des A Plaques nachgewiesen werden. Nachfolgende Analysen zeigten, dass Hirngefäße häufig mit A Plaques verbunden sind, was eine mögliche Assoziation von LTCCs mit A Plaques vermuten lässt. In weiteren Untersuchungen mit organotypischen Hirnschnitten konnte festgestellt werden, dass Substanz P und die CCBs Isradipin, Nimodipin, Nifedipin und Nicardipin Angiogenese in Blutgefäßen nahe von A Plaques induzieren können. Außerdem konnte belegt werden, dass A (Murines A42, humanes A42 und toxisches A25-35 Fragment) in der Lage ist, die mRNA Expression der LTCCs in primären wildtyp Astrozyten zu modulieren. Darüber hinaus konnte beobachtet werden, dass die CCBs Nifedipin und Nimodipin im Stande sind, neuroprotektiv auf dopaminerge Neurone in Axotomie-induzierten organotypischen Rattenhirnschnitten zu wirken. Zusammenfassend liefert diese Dissertation Einblicke in die komplexe Funktion reaktiver GFAP positiver Astrozyten und LTCCs, assoziiert mit A Plaques. Unsere Daten zeigen außerdem, dass LTCCs einen Ansatzpunkt für pharmakologische Therapien darstellen könnten, um einigen negativen Aspekten der AD entgegenzuwirken.Alzheimers disease (AD) is the most common neurodegenerative disorder in elderly people. The major hallmarks of this disease are (1) the extracellular accumulation of -amyloid (A) plaques, (2) the intracellular formation of hyperphosphorylated tau tangles, (3) synaptic loss resulting in cholinergic cell death and (4) the activation of glial cells such as astrocytes and microglia leading to inflammation. It is well-established that AD is frequently accompanied by the disruption of calcium homeostasis. Astrocytes surrounding A plaques are highly activated in an aged AD mouse model and possibly contribute to calcium dysfunction. The aims of this thesis were (1) to study the expression of L-type calcium channel (LTCC) subunits in reactive glial fibrillary acidic protein (GFAP) positive astroglia surrounding A plaques in an AD mouse model, (2) to analyze the mRNA expression profile of LTCC subunits in isolated primary mouse astrocytes and (3) to investigate the effects of calcium channel blockers (CCBs) on axotomized cholinergic and dopaminergic neurons in organotypic rat brain slices. In this thesis we could show that the upregulation of the LTCC subunit CaV1.2 1 in reactive astrocytes was associated with the development of A plaques in transgenic 11 month-old AD mice. Moreover, we found that the auxiliary subunit 4 was not colocalizing with reactive astrocytes. In-situ hybridization experiments showed that the mRNAs for CaV1.2 1, 2, 4 and 2-1 were predominantly expressed in the core area of the plaque, possibly representing a functional LTCC. Similarly, the mRNA for the neuropeptide preprotachykinin A, a precursor for substance P, was localized in the core. Confocal microscopy revealed that brain vessels were frequently associated with A plaques suggesting an association of LTCCs with A. Using organotypic brain slices we could show that substance P and the CCBs isradipine, nimodipine, nifedipine and nicardipine induced angiogenesis of vessels in close proximity to A plaques. Further, we could also show that A (murine A42, human A42 and toxic A25-35 fragment) is capable of modulating the mRNA expression of LTCCs in wild type primary astrocyte cultures. Moreover, we found that the CCBs nifedipine and nimodipine were capable of exerting neuroprotective effects on dopaminergic neurons in an axotomy-induced organotypic rat brain slice model. In summary, this thesis provides insights into the complex function of reactive GFAP positive astrocytes and LTCCs associated with A plaques. Our data also suggest that LTCCs could become possible pharmacological targets for counteracting some detrimental effects in AD.Nina DaschilEnth. u.a. 3 Veröff. d. Verf. aus den Jahren 2013 - 2014 . - Zsfassung in dt. SpracheInnsbruck, Med. Univ., Diss., 2015OeBB(VLID)33700

Organotypic vibrosections: Novel whole sagittal brain cultures

► Novel whole brain sagittal organotypic vibrosection in vitro model. ► Survival of cholinergic and dopaminergic neurons. ► Capillaries in whole brain sections in vitro. ► Long nerve fiber growth with neurotracing (Mini-Ruby). ► Striato-nigral varicosities