Scalable, cell type-selective, AAV-based in vivo CRISPR screening in the mouse brain.

CRISPR-based genetic screening directly in mammalian tissues in vivo is challenging due to the need for scalable, cell-type selective delivery and recovery of guide RNA libraries. We developed an in vivo adeno-associated virus-based and Cre recombinase-dependent workflow for cell type-selective CRISPR interference screening in mouse tissues. We demonstrate the power of this approach by identifying neuron-essential genes in the mouse brain using a library targeting over 2000 genes.

Identifying behavioral structure from deep variational embeddings of animal motion.

Quantification and detection of the hierarchical organization of behavior is a major challenge in neuroscience. Recent advances in markerless pose estimation enable the visualization of high-dimensional spatiotemporal behavioral dynamics of animal motion. However, robust and reliable technical approaches are needed to uncover underlying structure in these data and to segment behavior into discrete hierarchically organized motifs. Here, we present an unsupervised probabilistic deep learning framework that identifies behavioral structure from deep variational embeddings of animal motion (VAME). By using a mouse model of beta amyloidosis as a use case, we show that VAME not only identifies discrete behavioral motifs, but also captures a hierarchical representation of the motif's usage. The approach allows for the grouping of motifs into communities and the detection of differences in community-specific motif usage of individual mouse cohorts that were undetectable by human visual observation. Thus, we present a robust approach for the segmentation of animal motion that is applicable to a wide range of experimental setups, models and conditions without requiring supervised or a-priori human interference.

Novel App knock-in mouse model shows key features of amyloid pathology and reveals profound metabolic dysregulation of microglia.

BACKGROUND: Genetic mutations underlying familial Alzheimer's disease (AD) were identified decades ago, but the field is still in search of transformative therapies for patients. While mouse models based on overexpression of mutated transgenes have yielded key insights in mechanisms of disease, those models are subject to artifacts, including random genetic integration of the transgene, ectopic expression and non-physiological protein levels. The genetic engineering of novel mouse models using knock-in approaches addresses some of those limitations. With mounting evidence of the role played by microglia in AD, high-dimensional approaches to phenotype microglia in those models are critical to refine our understanding of the immune response in the brain. METHODS: We engineered a novel App knock-in mouse model (AppSAA) using homologous recombination to introduce three disease-causing coding mutations (Swedish, Arctic and Austrian) to the mouse App gene. Amyloid-ß pathology, neurodegeneration, glial responses, brain metabolism and behavioral phenotypes were characterized in heterozygous and homozygous AppSAA mice at different ages in brain and/ or biofluids. Wild type littermate mice were used as experimental controls. We used in situ imaging technologies to define the whole-brain distribution of amyloid plaques and compare it to other AD mouse models and human brain pathology. To further explore the microglial response to AD relevant pathology, we isolated microglia with fibrillar Aß content from the brain and performed transcriptomics and metabolomics analyses and in vivo brain imaging to measure energy metabolism and microglial response. Finally, we also characterized the mice in various behavioral assays. RESULTS: Leveraging multi-omics approaches, we discovered profound alteration of diverse lipids and metabolites as well as an exacerbated disease-associated transcriptomic response in microglia with high intracellular Aß content. The AppSAA knock-in mouse model recapitulates key pathological features of AD such as a progressive accumulation of parenchymal amyloid plaques and vascular amyloid deposits, altered astroglial and microglial responses and elevation of CSF markers of neurodegeneration. Those observations were associated with increased TSPO and FDG-PET brain signals and a hyperactivity phenotype as the animals aged. DISCUSSION: Our findings demonstrate that fibrillar Aß in microglia is associated with lipid dyshomeostasis consistent with lysosomal dysfunction and foam cell phenotypes as well as profound immuno-metabolic perturbations, opening new avenues to further investigate metabolic pathways at play in microglia responding to AD-relevant pathogenesis. The in-depth characterization of pathological hallmarks of AD in this novel and open-access mouse model should serve as a resource for the scientific community to investigate disease-relevant biology.

What electrophysiology tells us about Alzheimer's disease: a window into the synchronization and connectivity of brain neurons.

Electrophysiology provides a real-time readout of neural functions and network capability in different brain states, on temporal (fractions of milliseconds) and spatial (micro, meso, and macro) scales unmet by other methodologies. However, current international guidelines do not endorse the use of electroencephalographic (EEG)/magnetoencephalographic (MEG) biomarkers in clinical trials performed in patients with Alzheimer's disease (AD), despite a surge in recent validated evidence. This position paper of the ISTAART Electrophysiology Professional Interest Area endorses consolidated and translational electrophysiological techniques applied to both experimental animal models of AD and patients, to probe the effects of AD neuropathology (i.e., brain amyloidosis, tauopathy, and neurodegeneration) on neurophysiological mechanisms underpinning neural excitation/inhibition and neurotransmission as well as brain network dynamics, synchronization, and functional connectivity, reflecting thalamocortical and corticocortical residual capacity. Converging evidence shows relationships between abnormalities in EEG/MEG markers and cognitive deficits in groups of AD patients at different disease stages. The supporting evidence for the application of electrophysiology in AD clinical research as well as drug discovery pathways warrants an international initiative to include the use of EEG/MEG biomarkers in the main multicentric projects planned in AD patients, to produce conclusive findings challenging the present regulatory requirements and guidelines for AD studies.

Ovarian Cycle Stages Modulate Alzheimer-Related Cognitive and Brain Network Alterations in Female Mice.

Alzheimer's disease (AD) begins several decades before the onset of clinical symptoms, at a time when women may still undergo reproductive cycling. Whether ovarian functions alter substrates of AD pathogenesis is unknown. Here we show that ovarian cycle stages significantly modulate AD-related alterations in neural network patterns, cognitive impairments, and pathogenic protein production in the hAPP-J20 mouse model of AD. Female hAPP mice spent more time in estrogen-dominant cycle stages and these ovarian stages worsened AD-related network dysfunction and cognitive impairments. In contrast, progesterone-dominant stages and gonadectomy attenuated these AD-related deficits. Further studies revealed a direct role for estradiol in stimulating neural network excitability and susceptibility to seizures in hAPP mice and increasing amyloid beta levels. Understanding dynamic effects of the ovarian cycle on the female nervous system in disease, including AD, is of critical importance and may differ from effects on a healthy brain. The pattern of ovarian cycle effects on disease-related networks, cognition, and pathogenic protein expression may be relevant to young women at risk for AD.

Amyloid-ß/Fyn-induced synaptic, network, and cognitive impairments depend on tau levels in multiple mouse models of Alzheimer's disease.

Alzheimer's disease (AD), the most common neurodegenerative disorder, is a growing public health problem and still lacks effective treatments. Recent evidence suggests that microtubule-associated protein tau may mediate amyloid-ß peptide (Aß) toxicity by modulating the tyrosine kinase Fyn. We showed previously that tau reduction prevents, and Fyn overexpression exacerbates, cognitive deficits in human amyloid precursor protein (hAPP) transgenic mice overexpressing Aß. However, the mechanisms by which Aß, tau, and Fyn cooperate in AD-related pathogenesis remain to be fully elucidated. Here we examined the synaptic and network effects of this pathogenic triad. Tau reduction prevented cognitive decline induced by synergistic effects of Aß and Fyn. Tau reduction also prevented synaptic transmission and plasticity deficits in hAPP mice. Using electroencephalography to examine network effects, we found that tau reduction prevented spontaneous epileptiform activity in multiple lines of hAPP mice. Tau reduction also reduced the severity of spontaneous and chemically induced seizures in mice overexpressing both Aß and Fyn. To better understand these protective effects, we recorded whole-cell currents in acute hippocampal slices from hAPP mice with and without tau. hAPP mice with tau had increased spontaneous and evoked excitatory currents, reduced inhibitory currents, and NMDA receptor dysfunction. Tau reduction increased inhibitory currents and normalized excitation/inhibition balance and NMDA receptor-mediated currents in hAPP mice. Our results indicate that Aß, tau, and Fyn jointly impair synaptic and network function and suggest that disrupting the copathogenic relationship between these factors could be of therapeutic benefit.

Fyn kinase induces synaptic and cognitive impairments in a transgenic mouse model of Alzheimer's disease.

Human amyloid precursor protein (hAPP) transgenic mice with high levels of amyloid-beta (Abeta) develop behavioral deficits that correlate with the depletion of synaptic activity-related proteins in the dentate gyrus. The tyrosine kinase Fyn is altered in Alzheimer's disease brains and modulates premature mortality and synaptotoxicity in hAPP mice. To determine whether Fyn also modulates Abeta-induced behavioral deficits and depletions of synaptic activity-dependent proteins, we overexpressed Fyn in neurons of hAPP mice with moderate levels of Abeta production. Compared with nontransgenic controls and singly transgenic mice expressing hAPP or FYN alone, doubly transgenic FYN/hAPP mice had striking depletions of calbindin, Fos, and phosphorylated ERK (extracellular signal-regulated kinase), impaired neuronal induction of Arc, and impaired spatial memory retention. These deficits were qualitatively and quantitatively similar to those otherwise seen only in hAPP mice with higher Abeta levels. Surprisingly, levels of active Fyn were lower in high expresser hAPP mice than in NTG controls and lower in FYN/hAPP mice than in FYN mice. Suppression of Fyn activity may result from dephosphorylation by striatal-enriched phosphatase, which was upregulated in FYN/hAPP mice and in hAPP mice with high levels of Abeta. Thus, increased Fyn expression is sufficient to trigger prominent neuronal deficits in the context of even relatively moderate Abeta levels, and inhibition of Fyn activity may help counteract Abeta-induced impairments.

Aggressive amyloidosis in mice expressing human amyloid peptides with the Arctic mutation.

The Arctic mutation within the amyloid-beta (Abeta) peptide causes Alzheimer disease. In vitro, Arctic-mutant Abeta forms (proto)fibrils more effectively than wild-type Abeta. We generated transgenic mouse lines expressing Arctic-mutant human amyloid precursor proteins (hAPP). Amyloid plaques formed faster and were more extensive in Arctic mice than in hAPP mice expressing wild-type Abeta, even though Arctic mice had lower Abeta(1-42/1-40) ratios. Thus, the Arctic mutation is highly amyloidogenic in vivo.

Fyn kinase modulates synaptotoxicity, but not aberrant sprouting, in human amyloid precursor protein transgenic mice.

Alzheimer's disease (AD), the most common neurodegenerative disorder, results in progressive degeneration of synapses and aberrant sprouting of axon terminals. The mechanisms underlying these seemingly opposing cellular phenomena are unclear. We hypothesized that Fyn kinase may play a role in one or both of these processes because it is increased in AD brains and because it is involved in synaptic plasticity and axonal outgrowth. We investigated the effects of Fyn on AD-related synaptotoxicity and aberrant axonal sprouting by ablating or overexpressing Fyn in human amyloid precursor protein (hAPP) transgenic mice. On the fyn+/+ background, hAPP/amyloid beta peptide (Abeta) decreased hippocampal levels of synaptophysin-immunoreactive presynaptic terminals (SIPTs), consistent with previous findings. On the fyn-/- background, hAPP/Abeta did not affect SIPTs. SIPT reductions correlated with hippocampal Abeta levels in hAPP/fyn+/+, but not hAPP/fyn-/-, mice suggesting that Fyn provides a critical link between hAPP/Abeta and SIPTs. Furthermore, overexpression of Fyn exacerbated SIPT reductions in hAPP mice. We also found that the susceptibility of mice to hAPP/Abeta-induced premature mortality was decreased by Fyn ablation and increased by Fyn overexpression. In contrast, axonal sprouting in the hippocampus of hAPP mice was unaffected. We conclude that Fyn-dependent pathways are critical in AD-related synaptotoxicity and that the pathogenesis of hAPP/Abeta-induced neuronal alterations may be mechanistically heterogenous.

Neuronal depletion of calcium-dependent proteins in the dentate gyrus is tightly linked to Alzheimer's disease-related cognitive deficits.

Transgenic mice expressing human amyloid precursor proteins (hAPP) and amyloid-beta peptides (Abeta) in neurons develop phenotypic alterations resembling Alzheimer's disease (AD). The mechanisms underlying cognitive deficits in AD and hAPP mice are largely unknown. We have identified two molecular alterations that accurately reflect AD-related cognitive impairments. Learning deficits in mice expressing familial AD-mutant hAPP correlated strongly with decreased levels of the calcium-binding protein calbindin-D28k (CB) and the calcium-dependent immediate early gene product c-Fos in granule cells of the dentate gyrus, a brain region critically involved in learning and memory. These molecular alterations were age-dependent and correlated with the relative abundance of Abeta1-42 but not with the amount of Abeta deposited in amyloid plaques. CB reductions in the dentate gyrus primarily reflected a decrease in neuronal CB levels rather than a loss of CB-producing neurons. CB levels were also markedly reduced in granule cells of humans with AD, even though these neurons are relatively resistant to AD-related cell death. Thus, neuronal populations resisting cell death in AD and hAPP mice can still be drastically altered at the molecular level. The tight link between Abeta-induced cognitive deficits and neuronal depletion of CB and c-Fos suggests an involvement of calcium-dependent pathways in AD-related cognitive decline and could facilitate the preclinical evaluation of novel AD treatments.