Pregabalin improves axon regeneration and motor outcome in a rodent stroke model.

Ischaemic stroke remains a leading cause of death and disability worldwide. Surviving neurons in the peri-infarct area are able to establish novel axonal projections to juxtalesional regions, but this regeneration is curtailed by a growth-inhibitory environment induced by cells such as reactive astrocytes in the glial scar. Here, we found that the astroglial synaptogenic cue thrombospondin-1 is upregulated in the peri-infarct area, and hence tested the effects of the anticonvulsant pregabalin, a blocker of the neuronal thrombospondin-1 receptor Alpha2delta1/2, in a mouse model of cortical stroke. Studying axonal projections after cortical stroke in mice by three-dimensional imaging of cleared whole-brain preparations, we found that pregabalin, when administered systemically for 5 weeks after stroke, augments novel peri-infarct motor cortex projections and improves skilled forelimb motor function. Thus, the promotion of axon elongation across the glial scar by pregabalin represents a promising target beyond the acute phase after stroke to improve structural and functional recovery.

The development and physiological and pathophysiological functions of resident macrophages and glial cells.

In the past, brain function and the onset and progression of neurological diseases have been studied in a neuron-centric manner. However, in recent years the focus of many neuroscientists has shifted to other cell types that promote neurodevelopment and contribute to the functionality of neuronal networks in health and disease. Particularly microglia and astrocytes have been implicated in actively contributing to and controlling neuronal development, neuroinflammation, and neurodegeneration. Here, we summarize the development of brain-resident macrophages and astrocytes and their core functions in the developing brain. We discuss their contribution and intercellular crosstalk during tissue homeostasis and pathophysiology. We argue that in-depth knowledge of non-neuronal cells in the brain could provide novel therapeutic targets to reverse or contain neurological diseases.

Creld2 function during unfolded protein response is essential for liver metabolism homeostasis.

The unfolded protein response (UPR) is associated with hepatic metabolic function, yet it is not well understood how endoplasmic reticulum (ER) disturbance might influence metabolic homeostasis. Here, we describe the physiological function of Cysteine-rich with EGF-like domains 2 (Creld2), previously characterized as a downstream target of the ER-stress signal transducer Atf6. To this end, we generated Creld2-deficient mice and induced UPR by injection of tunicamycin. Creld2 augments protein folding and creates an interlink between the UPR axes through its interaction with proteins involved in the cellular stress response. Thereby, Creld2 promotes tolerance to ER stress and recovery from acute stress. Creld2-deficiency leads to a dysregulated UPR and causes the development of hepatic steatosis during ER stress conditions. Moreover, Creld2-dependent enhancement of the UPR assists in the regulation of energy expenditure. Furthermore, we observed a sex dimorphism in human and mouse livers with only male patients showing an accumulation of CRELD2 protein during the progression from non-alcoholic fatty liver disease to non-alcoholic steatohepatitis and only male Creld2-deficient mice developing hepatic steatosis upon aging. These results reveal a Creld2 function at the intersection between UPR and metabolic homeostasis and suggest a mechanism in which chronic ER stress underlies fatty liver disease in males.

Slc1a3-2A-CreERT2 mice reveal unique features of Bergmann glia and augment a growing collection of Cre drivers and effectors in the 129S4 genetic background.

Genetic variation is a primary determinant of phenotypic diversity. In laboratory mice, genetic variation can be a serious experimental confounder, and thus minimized through inbreeding. However, generalizations of results obtained with inbred strains must be made with caution, especially when working with complex phenotypes and disease models. Here we compared behavioral characteristics of C57Bl/6-the strain most widely used in biomedical research-with those of 129S4. In contrast to 129S4, C57Bl/6 demonstrated high within-strain and intra-litter behavioral hyperactivity. Although high consistency would be advantageous, the majority of disease models and transgenic tools are in C57Bl/6. We recently established six Cre driver lines and two Cre effector lines in 129S4. To augment this collection, we genetically engineered a Cre line to study astrocytes in 129S4. It was validated with two Cre effector lines: calcium indicator gCaMP5g-tdTomato and RiboTag-a tool widely used to study cell type-specific translatomes. These reporters are in different genomic loci, and in both the Cre was functional and astrocyte-specific. We found that calcium signals lasted longer and had a higher amplitude in cortical compared to hippocampal astrocytes, genes linked to a single neurodegenerative disease have highly divergent expression patterns, and that ribosome proteins are non-uniformly expressed across brain regions and cell types.

Inhibition of Stat3-mediated astrogliosis ameliorates pathology in an Alzheimer's disease model.

Reactive astrogliosis is a hallmark of Alzheimer's disease (AD), but its role for disease initiation and progression has remained incompletely understood. We here show that the transcription factor Stat3 (signal transducer and activator of transcription 3), a canonical inducer of astrogliosis, is activated in an AD mouse model and human AD Therefore, using a conditional knockout approach, we deleted Stat3 specifically in astrocytes in the APP/PS1 model of AD We found that Stat3-deficient APP/PS1 mice show decreased ß-amyloid levels and plaque burden. Plaque-close microglia displayed a more complex morphology, internalized more ß-amyloid, and upregulated amyloid clearance pathways in Stat3-deficient mice. Moreover, astrocyte-specific Stat3-deficient APP/PS1 mice showed decreased pro-inflammatory cytokine activation and lower dystrophic neurite burden, and were largely protected from cerebral network imbalance. Finally, Stat3 deletion in astrocytes also strongly ameliorated spatial learning and memory decline in APP/PS1 mice. Importantly, these protective effects on network dysfunction and cognition were recapitulated in APP/PS1 mice systemically treated with a preclinical Stat3 inhibitor drug. In summary, our data implicate Stat3-mediated astrogliosis as an important therapeutic target in AD.

Stroke target identification guided by astrocyte transcriptome analysis.

Astrocytes support normal brain function, but may also contribute to neurodegeneration when they become reactive under pathological conditions such as stroke. However, the molecular underpinnings of this context-dependent interplay between beneficial and detrimental properties in reactive astrogliosis have remained incompletely understood. Therefore, using the RiboTag technique, we immunopurified translating mRNAs specifically from astrocytes 72 hr after transient middle cerebral artery occlusion in mice (tMCAO), thereby generating a stroke-specific astroglial translatome database. We found that compared to control brains, reactive astrocytes after tMCAO show an enrichment of transcripts linked to the A2 phenotype, which has been associated with neuroprotection. However, we found that astrocytes also upregulate a large number of potentially neurotoxic genes. In total, we identified the differential expression of 1,003 genes and 38 transcription factors, of which Stat3, Sp1, and Spi1 were the most prominent. To further explore the effects of Stat3-mediated pathways on stroke pathogenesis, we subjected mice with an astrocyte-specific conditional deletion of Stat3 to tMCAO, and found that these mice have reduced stroke volume and improved motor outcome 72 hr after focal ischemia. Taken together, our study extends the emerging database of novel astrocyte-specific targets for stroke therapy, and supports the role of astrocytes as critical safeguards of brain function in health and disease.

P2Y1 receptor blockade normalizes network dysfunction and cognition in an Alzheimer's disease model.

Astrocytic hyperactivity is an important contributor to neuronal-glial network dysfunction in Alzheimer's disease (AD). We have previously shown that astrocyte hyperactivity is mediated by signaling through the P2Y1 purinoreceptor (P2Y1R) pathway. Using the APPPS1 mouse model of AD, we here find that chronic intracerebroventricular infusion of P2Y1R inhibitors normalizes astroglial and neuronal network dysfunction, as measured by in vivo two-photon microscopy, augments structural synaptic integrity, and preserves hippocampal long-term potentiation. These effects occur independently from ß-amyloid metabolism or plaque burden but are associated with a higher morphological complexity of periplaque reactive astrocytes, as well as reduced dystrophic neurite burden and greater plaque compaction. Importantly, APPPS1 mice chronically treated with P2Y1R antagonists, as well as APPPS1 mice carrying an astrocyte-specific genetic deletion (Ip3r2-/-) of signaling pathways downstream of P2Y1R activation, are protected from the decline of spatial learning and memory. In summary, our study establishes the restoration of network homoeostasis by P2Y1R inhibition as a novel treatment target in AD.

Inhibition of the MID1 protein complex: a novel approach targeting APP protein synthesis.

Alzheimer's disease (AD) is characterized by two neuropathological hallmarks: senile plaques, which are composed of amyloid-ß (Aß) peptides, and neurofibrillary tangles, which are composed of hyperphosphorylated tau protein. Aß peptides are derived from sequential proteolytic cleavage of the amyloid precursor protein (APP). In this study, we identified a so far unknown mode of regulation of APP protein synthesis involving the MID1 protein complex: MID1 binds to and regulates the translation of APP mRNA. The underlying mode of action of MID1 involves the mTOR pathway. Thus, inhibition of the MID1 complex reduces the APP protein level in cultures of primary neurons. Based on this, we used one compound that we discovered previously to interfere with the MID1 complex, metformin, for in vivo experiments. Indeed, long-term treatment with metformin decreased APP protein expression levels and consequently Aß in an AD mouse model. Importantly, we have initiated the metformin treatment late in life, at a time-point where mice were in an already progressed state of the disease, and could observe an improved behavioral phenotype. These findings together with our previous observation, showing that inhibition of the MID1 complex by metformin also decreases tau phosphorylation, make the MID1 complex a particularly interesting drug target for treating AD.

Targeted inactivation of the mouse epididymal beta-defensin 41 alters sperm flagellar beat pattern and zona pellucida binding.

During epididymal maturation, sperm acquire the ability to swim progressively by interacting with proteins secreted by the epididymal epithelium. Beta-defensin proteins, expressed in the epididymis, continue to regulate sperm motility during capacitation and hyperactivation in the female reproductive tract. We characterized the mouse beta-defensin 41 (DEFB41), by generating a mouse model with iCre recombinase inserted into the first exon of the gene. The homozygous Defb41(iCre/iCre) knock-in mice lacked Defb41 expression and displayed iCre recombinase activity in the principal cells of the proximal epididymis. Heterozygous Defb41(iCre/+) mice can be used to generate epididymis specific conditional knock-out mouse models. Homozygous Defb41(iCre/iCre) sperm displayed a defect in sperm motility with the flagella primarily bending in the pro-hook conformation while capacitated wild-type sperm more often displayed the anti-hook conformation. This led to a reduced straight line motility of Defb41(iCre/iCre) sperm and weaker binding to the oocyte. Thus, DEFB41 is required for proper sperm maturation.