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Neurons are highly polarized cells that extend a single axon and several dendrites. Studies with cultured neurons indicate that the proximal portion of the axon, denoted as the axon initial segment (AIS), maintains neuronal polarity in vitro. The membrane-adaptor protein ankyrinG (ankG) is an essential component of the AIS. To determine the relevance of ankG for neuronal polarity in vivo, we studied mice with a cerebellum-specific ankG deficiency. Strikingly, ankG-depleted axons develop protrusions closely resembling dendritic spines. Such axonal spines are enriched with postsynaptic proteins, including ProSAP1/Shank2 and ionotropic and metabotropic glutamate receptors. In addition, immunofluorescence indicated that axonal spines are contacted by presynaptic glutamatergic boutons. For further analysis, double mutants were obtained by crossbreeding ankG⁻/⁻ mice with L7/Purkinje cell-specific promoter 2 (PCP2) mice expressing enhanced green fluorescent protein (EGFP) in Purkinje cells (PCs). This approach allowed precise confocal microscopic mapping of EGFP-positive spiny axons and their subsequent identification at the electron microscopic level. Ultrastructurally, axonal spines contained a typical postsynaptic density and established asymmetric excitatory synapses with presynaptic boutons containing synaptic vesicles. In the shaft of spiny axons, typical ultrastructural features of the AIS, including the membrane-associated dense undercoating and cytoplasmic bundles of microtubules, were absent. Finally, using time-lapse imaging of organotypic cerebellar slice cultures, we demonstrate that nonspiny PC axons of EGFP-positive/ankG⁻/⁻ mice acquire a spiny phenotype within a time range of only 3 days. Collectively, these findings demonstrate that axons of ankG-deficient mice acquire hallmark features of dendrites. AnkG thus is important for maintaining appropriate axo-dendritic polarity in vivo.

The disintegrin and metalloproteinases ADAM10 and ADAM17 are regarded as the most important α-secretases involved in the physiological processing of amyloid precursor protein (APP) in brain. Since it has been suggested that processing of APP by α-secretases could be involved in the reorganization of the brain following injury, we studied mRNA expression of the two α-secretases Adam10 and Adam17, the ß-secretase Bace1, and the App-gene family (App, Aplp1, Aplp2) in the dentate gyrus of the mouse following entorhinal denervation. Using laser microdissection, tissue was harvested from the outer molecular layer and the granule cell layer of the denervated dentate gyrus. Expression levels of candidate genes were assessed using Affymetrix GeneChip Mouse Gene 1.0 ST arrays and reverse transcription-quantitative PCR, revealing an upregulation of Adam10 mRNA and Adam17 mRNA in the denervated outer molecular layer and an upregulation of Adam10 mRNA and App mRNA in the dentate granule cell layer. Immunolabeling for ADAM10 or ADAM17 in combination with markers for astro- and microglia revealed an increased labeling of ADAM10 and ADAM17 in the denervated outer molecular layer that was associated with reactive astrocytes but not with microglia. Collectively, these data show that denervation affects the expression level of APP and its two most important α-secretases. This suggests that APP-processing could be shifted towards the non-amyloidogenic pathway in denervated areas of the brain and, thus, towards the formation of neuroprotective APP cleavage products, such as APPsα.

Mechanisms behind how the immune system signals to the brain in response to systemic inflammation are not fully understood. Transgenic mice expressing Cre recombinase specifically in the hematopoietic lineage in a Cre reporter background display recombination and marker gene expression in Purkinje neurons. Here we show that reportergene expression in neurons is caused by intercellular transfer of functional Cre recombinase messenger RNA from immune cells into neurons in the absence of cell fusion. In vitro purified secreted extracellular vesicles (EVs) from blood cells contain Cre mRNA, which induces recombination in neurons when injected into the brain. Although Cre-mediated recombination events in the brain occur very rarely in healthy animals, their number increases considerably in different injury models, particularly under inflammatory conditions, and extend beyond Purkinje neurons to other neuronal populations in cortex, hippocampus, and substantia nigra. Recombined Purkinje neurons differ in their miRNA profile from their nonrecombined counterparts, indicating physiological significance. These observations reveal the existence of a previously unrecognized mechanism to communicate RNA-based signals between the hematopoietic system and various organs, including the brain, in response to inflammation.

Aggregates of medin amyloid (a fragment of the protein MFG-E8, also known as lactadherin) are found in the vasculature of almost all humans over 50 years of age 1 , 2 , making it the most common amyloid currently known. We recently reported that medin also aggregates in blood vessels of ageing wild-type mice, causing cerebrovascular dysfunction 3 . Here we demonstrate in amyloid-β precursor protein (APP) transgenic mice and in patients with Alzheimer’s disease that medin co-localizes with vascular amyloid-β deposits, and that in mice, medin deficiency reduces vascular amyloid-β deposition by half. Moreover, in both the mouse and human brain, MFG-E8 is highly enriched in the vasculature and both MFG-E8 and medin levels increase with the severity of vascular amyloid-β burden. Additionally, analysing data from 566 individuals in the ROSMAP cohort, we find that patients with Alzheimer’s disease have higher MFGE8 expression levels, which are attributable to vascular cells and are associated with increased measures of cognitive decline, independent of plaque and tau pathology. Mechanistically, we demonstrate that medin interacts directly with amyloid-β to promote its aggregation, as medin forms heterologous fibrils with amyloid-β, affects amyloid-β fibril structure, and cross-seeds amyloid-β aggregation both in vitro and in vivo. Thus, medin could be a therapeutic target for prevention of vascular damage and cognitive decline resulting from amyloid-β deposition in the blood vessels of the brain. Medin promotes the formation of vascular aggregates with amyloid-β in mouse models and in human patients with Alzheimer’s disease, and is associated with vascular defects and cognitive decline.

Quantitative PCR (qPCR) is a widely used method to study gene expression changes following brain injury. The accuracy of this method depends on the tissue harvested, the time course analyzed and, in particular on the choice of appropriate internal controls, i.e., reference genes (RGs). In the present study we have developed and validated an algorithm for the accurate normalization of qPCR data using laser microdissected tissue from the mouse dentate gyrus after entorhinal denervation at 0, 1, 3, 7, 14 and 28 days postlesion. The expression stabilities of ten candidate RGs were evaluated in the denervated granule cell layer (gcl) and outer molecular layer (oml) of the dentate gyrus. Advanced software algorithms demonstrated differences in stability for single RGs in the two layers at several time points postlesion. In comparison, a normalization index of several stable RGs covered the entire post-lesional time course and showed high stability. Using these RGs, we validated our findings and quantified glial fibrillary acidic protein ( Gfap ) mRNA and allograft inflammatory factor 1 ( Aif1/Iba1 ) mRNA in the denervated oml. We compared the use of single RGs for normalization with the normalization index and found that single RGs yield variable results. In contrast, the normalization index gave stable results. In sum, our study shows that qPCR can yield precise, reliable, and reproducible datasets even under such complex conditions as brain injury or denervation, provided appropriate RGs for the model are used. The algorithm reported here can easily be adapted and transferred to any other brain injury model.

Large spines are stable and important for memory trace formation. The majority of large spines also contains synaptopodin (SP), an actin-modulating and plasticity-related protein. Since SP stabilizes F-actin, we speculated that the presence of SP within large spines could explain their long lifetime. Indeed, using 2-photon time-lapse imaging of SP-transgenic granule cells in mouse organotypic tissue cultures we found that spines containing SP survived considerably longer than spines of equal size without SP. Of note, SP-positive (SP+) spines that underwent pruning first lost SP before disappearing. Whereas the survival time courses of SP+ spines followed conditional two-stage decay functions, SP-negative (SP-) spines and all spines of SP-deficient animals showed single-phase exponential decays. This was also the case following afferent denervation. These results implicate SP as a major regulator of long-term spine stability: SP clusters stabilize spines, and the presence of SP indicates spines of high stability.

Medin is the most common amyloid known in humans, as it can be found in blood vessels of the upper body in virtually everybody over 50 years of age. However, it remains unknown whether deposition of Medin plays a causal role in age-related vascular dysfunction. We now report that aggregates of Medin also develop in the aorta and brain vasculature of wild-type mice in an age-dependent manner. Strikingly, genetic deficiency of the Medin precursor protein, MFG-E8, eliminates not only vascular aggregates but also prevents age-associated decline of cerebrovascular function in mice. Given the prevalence of Medin aggregates in the general population and its role in vascular dysfunction with aging, targeting Medin may become a novel approach to sustain healthy aging.

The cation-chloride cotransporters KCC2 and NKCC1 regulate the intracellular Cl − concentration and cell volume of neurons and/or glia. The Cl − extruder KCC2 is expressed at higher levels than the Cl − transporter NKCC1 in mature compared to immature neurons, accounting for the developmental shift from high to low Cl − concentration and from depolarizing to hyperpolarizing currents through GABA-A receptors. Previous studies have shown that KCC2 expression is downregulated following central nervous system injury, returning neurons to a more excitable state, which can be pathological or adaptive. Here, we show that deafferentation of the dendritic segments of granule cells in the outer (oml) and middle (mml) molecular layer of the dentate gyrus via entorhinal denervation in vivo leads to cell-type- and layer-specific changes in the expression of KCC2 and NKCC1. Microarray analysis validated by reverse transcription-quantitative polymerase chain reaction revealed a significant decrease in Kcc2 mRNA in the granule cell layer 7 days post-lesion. In contrast, Nkcc1 mRNA was upregulated in the oml/mml at this time point. Immunostaining revealed a selective reduction in KCC2 protein expression in the denervated dendrites of granule cells and an increase in NKCC1 expression in reactive astrocytes in the oml/mml. The NKCC1 upregulation is likely related to the increased activity of astrocytes and/or microglia in the deafferented region, while the transient KCC2 downregulation in granule cells may be associated with denervation-induced spine loss, potentially also serving a homeostatic role via boosting GABAergic depolarization. Furthermore, the delayed KCC2 recovery might be involved in the subsequent compensatory spinogenesis.

Sprouting of surviving axons is one of the major reorganization mechanisms of the injured brain contributing to a partial restoration of function. Of note, sprouting is maturation as well as age-dependent and strong in juvenile brains, moderate in adult and weak in aged brains. We have established a model system of complex organotypic tissue cultures to study sprouting in the dentate gyrus following entorhinal denervation. Entorhinal denervation performed after 2 weeks postnatally resulted in a robust, rapid, and very extensive sprouting response of commissural/associational fibers, which could be visualized using calretinin as an axonal marker. In the present study, we analyzed the effect of maturation on this form of sprouting and compared cultures denervated at 2 weeks postnatally with cultures denervated at 4 weeks postnatally. Calretinin immunofluorescence labeling as well as time-lapse imaging of virally-labeled (AAV2-hSyn1-GFP) commissural axons was employed to study the sprouting response in aged cultures. Compared to the young cultures commissural/associational sprouting was attenuated and showed a pattern similar to the one following entorhinal denervation in adult animals in vivo . We conclude that a maturation-dependent attenuation of sprouting occurs also in vitro , which now offers the chance to study, understand and influence maturation-dependent differences in brain repair in these culture preparations.

Collateral sprouting of surviving axons contributes to the synaptic reorganization after brain injury. To study this clinically relevant phenomenon, we used complex organotypic tissue cultures of mouse entorhinal cortex (EC) and hippocampus (H). Single EC-H cultures were generated to analyze associational sprouting, and double EC-H cultures were used to evaluate commissural sprouting of mossy cells in the dentate gyrus (DG) following entorhinal denervation. Entorhinal denervation (transection of the perforant path) was performed at 14 days (DIV) and associational/commissural sprouting was assessed at 28 DIV. First, associational sprouting was studied in genetically hybrid EC-H cultures of beta-actin-GFPtg and wild-type mice. Using calretinin as a marker, associational axons were found to re-innervate almost the entire entorhinal target zone. Denervation experiments performed with EC-H cultures of Thy1-YFPtg mice, in which mossy cells are YFP-positive, confirmed that the overwhelming majority of sprouting associational calretinin-positive axons are mossy cell axons. Second, we analyzed associational/commissural sprouting by combining wild-type EC-H cultures with calretinin-deficient EC-H cultures. In these cultures, only wild-type mossy cells contain calretinin, and associational and commissural mossy cell collaterals can be distinguished using calretinin as a marker. Nearly the entire DG entorhinal target zone was re-innervated by sprouting of associational and commissural mossy cell axons. Finally, viral labeling of newly formed associational/commissural axons revealed a rapid post-lesional sprouting response. These findings demonstrate extensive and rapid re-innervation of the denervated DG outer molecular layer by associational and commissural mossy cell axons, similar to what has been reported to occur in juvenile rodent DG .