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Intron retention (IR) has been implicated in the pathogenesis of complex diseases such as cancers; its association with Alzheimer's disease (AD) remains unexplored. We performed genome‐wide analysis of IR through integrating genetic, transcriptomic, and proteomic data of AD subjects and mouse models from the Accelerating Medicines Partnership‐Alzheimer's Disease project. We identified 4535 and 4086 IR events in 2173 human and 1736 mouse genes, respectively. Quantitation of IR enabled the identification of differentially expressed genes that conventional exon‐level approaches did not reveal. There were significant correlations of intron expression within innate immune genes, like HMBOX1, with AD in humans. Peptides with a high probability of translation from intron‐retained mRNAs were identified using mass spectrometry. Further, we established AD‐specific intron expression Quantitative Trait Loci, and identified splicing‐related genes that may regulate IR. Our analysis provides a novel resource for the search for new AD biomarkers and pathological mechanisms.

Immunotherapies designed to treat neurodegenerative tauopathies that primarily engage extracellular tau may have limited efficacy as tau is primarily intracellular. We generated tau-targeting single-chain variable fragments (scFvs) and intrabodies (iBs) from the phosphorylated tau-specific antibodies CP13 and PHF1 and the pan-tau antibody Tau5. Recombinant adeno-associated virus (rAAV) was utilized to express these antibody fragments in homozygous JNPL3 P301L tau mice. Two iBs (CP13i, PHF1i) and one scFv (PHF1s) abrogated tau pathology and delayed time to severe hindlimb paralysis. In a second tauopathy model (rTg4510), CP13i and PHF1i reduced tau pathology, but cognate scFvs did not. These data demonstrate that (1) disease-modifying efficacy does not require antibody effector functions, (2) the intracellular targeting of tau with phosphorylated tau-specific iBs is more effective than extracellular targeting with the scFvs, and (3) robust effects on tau pathology before neurodegeneration only resulted in modest disease modification as assessed by delay of severe motor phenotype., Graphical Abstract, Tau-specific antibodies have been shown to reduce tau pathology in mice by binding extracellular tau. Goodwin et al. developed phosphorylated tau-specific single-chain variable fragments expressed in the cytoplasm and targeted to bind intracellular tau. These intrabodies reduced pathology and slowed the progression of tau-induced neurodegeneration in transgenic mice.

The impact of oral microbial dysbiosis on Alzheimer’s disease (AD) remains controversial. Building off recent studies reporting that various microbes might directly seed or promote amyloid β (Aβ) deposition, we evaluated the effects of periodontal bacteria (Porphyromonas gingivalis, Treponema denticola) and supragingival commensal (Streptococcus gordonii) oral bacterial infection in the APP-transgenic CRND8 (Tg) mice model of AD. We tracked bacterial colonization and dissemination, and monitored effects on gliosis and amyloid deposition. Chronic oral infection did not accelerate Aβ deposition in Tg mice but did induce alveolar bone resorption, IgG immune response, and an intracerebral astrogliosis (GFAP: glial fibrillary acidic protein). In contrast, intracerebral inoculation of live but not heat-killed P. gingivalis increased Aβ deposition and Iba-1 (ionized calcium-binding adaptor-1) microgliosis after 8 weeks of bacterial infection but not at 4 days. These data show that there may be differential effects of infectious microbes on glial activation and amyloid deposition depending on the species and route of inoculation, and thereby provide an important framework for future studies. Indeed, these studies demonstrate marked effects on amyloid β deposition only in a fairly non-physiologic setting where live bacteria is injected directly into the brain.

A high-affinity monoclonal antibody (CTRND05) targeting corticotropin-releasing factor (CRF) blocks stress-induced corticosterone increases, counteracts effects of chronic variable stress, and induces other phenotypes consistent with suppression of the HPA axis., Hypothalamic–pituitary–adrenal (HPA) axis dysfunction contributes to numerous human diseases and disorders. We developed a high-affinity monoclonal antibody, CTRND05, targeting corticotropin-releasing factor (CRF). In mice, CTRND05 blocks stress-induced corticosterone increases, counteracts effects of chronic variable stress, and induces other phenotypes consistent with suppression of the HPA axis. CTRND05 induces skeletal muscle hypertrophy and increases lean body mass, effects not previously reported with small-molecule HPA-targeting pharmacologic agents. Multiorgan transcriptomics demonstrates broad HPA axis target engagement through altering levels of known HPA-responsive transcripts such as Fkbp5 and Myostatin and reveals novel HPA-responsive pathways such as the Apelin-Apelin receptor system. These studies demonstrate the therapeutic potential of CTRND05 as a suppressor of the HPA axis and serve as an exemplar of a potentially broader approach to target neuropeptides with immunotherapies, as both pharmacologic tools and novel therapeutics.

SUMMARY We present a consensus atlas of the human brain transcriptome in Alzheimer’s disease (AD), based on meta-analysis of differential gene expression in 2,114 postmortem samples. We discover 30 brain coexpression modules from seven regions as the major source of AD transcriptional perturbations. We next examine overlap with 251 brain differentially expressed gene sets from mouse models of AD and other neurodegenerative disorders. Human-mouse overlaps highlight responses to amyloid versus tau pathology and reveal age- and sex-dependent expression signatures for disease progression. Human coexpression modules enriched for neuronal and/or microglial genes broadly overlap with mouse models of AD, Huntington’s disease, amyotrophic lateral sclerosis, and aging. Other human coexpression modules, including those implicated in proteostasis, are not activated in AD models but rather following other, unexpected genetic manipulations. Our results comprise a cross-species resource, highlighting transcriptional networks altered by human brain pathophysiology and identifying correspondences with mouse models for AD preclinical studies., Graphical Abstract, In Brief Wan et al. develop a consensus atlas of the human brain transcriptome in Alzheimer’s disease, based on 2,114 postmortem samples, and examine overlap with 251 expression signatures from mouse brain RNA sequencing experiments, including many neurodegenerative disease models.

Background Recombinant adeno-associated virus (rAAV) is widely used in the neuroscience field to manipulate gene expression in the nervous system. However, a limitation to the use of rAAV vectors is the time and expense needed to produce them. To overcome this limitation, we evaluated whether unpurified rAAV vectors secreted into the media following scalable PEI transfection of HEK293T cells can be used in lieu of purified rAAV. Methods We packaged rAAV2-EGFP vectors in 30 different wild-type and mutant capsids and subsequently collected the media containing secreted rAAV. Genomic titers of each rAAV vector were assessed and the ability of each unpurified virus to transduce primary mixed neuroglial cultures (PNGCs), organotypic brain slice cultures (BSCs) and the mouse brain was evaluated. Results There was ~ 40-fold wide variance in the average genomic titers of the rAAV2-EGFP vector packaged in the 30 different capsids, ranging from a low ~ 4.7 × 1010 vector genomes (vg)/mL for rAAV2/5-EGFP to a high of ~ 2.0 × 1012 vg/mL for a capsid mutant of rAAV2/8-EGFP. In PNGC studies, we observed a wide range of transduction efficiency among the 30 capsids evaluated, with the rAAV2/6-EGFP vector demonstrating the highest overall transduction efficiency. In BSC studies, we observed robust transduction by wild-type capsid vectors rAAV2/6, 2/8 and 2/9, and by capsid mutants of rAAV2/1, 2/6, and 2/8. In the in vivo somatic brain transgenesis (SBT) studies, we found that intra-cerebroventricular injection of media containing unpurified rAAV2-EGFP vectors packaged with select mutant capsids resulted in abundant EGFP positive neurons and astrocytes in the hippocampus and forebrain of non-transgenic mice. We demonstrate that unpurified rAAV can express transgenes at equivalent levels to lysate-purified rAAV both in vitro and in vivo. We also show that unpurified rAAV is sufficient to drive tau pathology in BSC and neuroinflammation in vivo, recapitulating previous studies using purified rAAV. Conclusions Unpurified rAAV vectors secreted into the media can efficiently transduce brain cells in vitro and in vivo, providing a cost-effective way to manipulate gene expression. The use of unpurified virus will greatly reduce costs of exploratory studies and further increase the utility of rAAV vectors for standard laboratory use.

SUMMARYHuman brain transcriptomes can highlight biological pathways associated with Alzheimer’s disease (AD); however, challenges remain to link expression changes with causal triggers. We have examined 30 AD-associated, gene coexpression modules from human brains for overlap with 251 differentially-expressed gene sets from mouse brain RNA-sequencing experiments, including from models of AD and other neurodegenerative disorders. Human-mouse overlaps highlight responses to amyloid versus neurofibrillary tangle pathology and further reveal age- and sex-dependent expression signatures for AD progression. Human coexpression modules enriched for neuronal and/or microglial genes overlap broadly with signatures from mouse models of AD, Huntington’s disease, Amyotrophic Lateral Sclerosis, and also aging. Several human AD coexpression modules, including those implicated in the unfolded protein response and oxidative phosphorylation, were not activated in AD models, but instead were detected following other, unexpected mouse genetic manipulations. Our results comprise a powerful, cross-species resource and pinpoint experimental models for diverse features of AD pathophysiology from human brain transcriptomes.

Phosphorylation of the microtubule associated protein tau is an important modulator of its normal physiological functioning; however, it may also contribute to tau mis-folding and aggregation in neurodegenerative diseases, which are collectively termed tauopathies. As such, the investigations of tau phosphorylation and kinases that modify tau are important in trying to elucidate tau function and the mechanisms involved in the development of tauopathies. We have recently demonstrated that the putative tau kinase leucine-rich repeat kinase 2 is capable of phosphorylating tau at threonines 169 and 175 in vitro, and it has been previously shown that hyperphosphorylation at threonine 175 occurs in filamentous tau species from Alzheimer's brain tissue. These prior findings suggest that further studies of phosphorylation of tau at these epitopes may shed light on the pathogenesis of tauopathies. There is, however, a lack of tools available to analyze phosphorylation of tau at these sites. This study aimed to bridge that resource gap by generating monoclonal antibodies against tau phosphorylated at either threonine 169 or 175. While we did not succeed in generating a phospho-specific antibody, we did generate an antibody, MHT2, which is specific for human tau encompassing the threonine 169/175 epitope region. Immunostaining of transgenic rTg4510 mouse tissue as well as human tauopathy cases with MHT2 indicates that this antibody selectively detects cytoplasmic tau in the form of neurofibrillary tangles, and that it may have a further specificity pertaining to severity of disease progression, either because of phosphorylation or conformational bias.

Low molecular weight oligomers of amyloid-β (Aβ) have emerged as the primary toxic agents in the etiology of Alzheimer disease (AD). Polymorphism observed within the aggregation end products of fibrils are known to arise due to microstructural differences among the oligomers. Diversity in aggregate morphology correlates with the differences in AD, cementing the idea that conformational strains of oligomers could be significant in phenotypic outcomes. Therefore, it is imperative to determine the ability of strains to faithfully propagate their structure. Here we report fibril propagation of an Aβ42 dodecamer called large fatty acid-derived oligomers (LFAOs). The LFAO oligomeric strain selectively induces acute cerebral amyloid angiopathy (CAA) in neonatally-injected transgenic CRND8 mice. Propagation in-vitro occurs as a three-step process involving the association of LFAO units. LFAO-seeded fibrils possess distinct morphology made of repeating LFAO units that could be regenerated upon sonication. Overall, these data bring forth an important mechanistic perspective into strain-specific propagation of oligomers that has remained elusive thus far.

Alzheimer's disease (AD) and familial Danish dementia (FDD) are degenerative neurological diseases characterized by amyloid pathology. Normal human sera contain IgG antibodies that specifically bind diverse preamyloid and amyloid proteins and have shown therapeutic potentialin vitroandin vivo. We cloned one of these antibodies, 3H3, from memory B cells of a healthy individual using a hybridoma method. 3H3 is an affinity-matured IgG that binds a pan-amyloid epitope, recognizing both Aβ and λ Ig light chain (LC) amyloids, which are associated with AD and primary amyloidosis, respectively. The pan-amyloid-binding properties of 3H3 were demonstrated using ELISA, immunohistochemical studies, and competition binding assays. Functional studies showed that 3H3 inhibits both Aβ and LC amyloid formationin vitroand abrogates disruption of hippocampal synaptic plasticity by AD-patient-derived soluble Aβin vivo. A 3H3 single-chain variable fragment (scFv) retained the binding specificity of the 3H3 IgG and, when expressed in the brains of transgenic mice using an adeno-associated virus (AAV) vector, decreased parenchymal Aβ amyloid deposition in TgCRND8 mice and ADan (Danish Amyloid) cerebral amyloid angiopathy in the mouse model of FDD. These data indicate that naturally occurring human IgGs can recognize a conformational, amyloid-specific epitope and have potent anti-amyloid activities, providing a rationale to test their potential as antibody therapeutics for diverse neurological and other amyloid diseases.

The architecture of the spinal cord makes efficient delivery of recombinant adeno-associated virus (rAAV) vectors throughout the neuraxis challenging. We describe a paradigm in which small amounts of virus delivered intraspinally to newborn mice result in robust rAAV-mediated transgene expression in the spinal cord. We compared the efficacy of rAAV2/1, 2/5, 2/8, and 2/9 encoding EGFP delivered to the hindlimb muscle (IM), cisterna magna (ICM), or lumbar spinal cord (IS) of neonatal pups. IS injection of all four capsids resulted in robust transduction of the spinal cord with rAAV2/5, 2/8, and 2/9 vectors appearing to be transported to brain. ICM injection resulted in widespread expression of EGFP in the brain, and upper spinal cord. IM injection resulted in robust muscle expression, with only rAAV2/8 and 2/9 transducing spinal motor and sensory neurons. As proof of concept, we use the IS paradigm to express murine Interleukin (IL)-10 in the spinal cord of the SOD1-G93A transgenic mouse model of amyotrophic lateral sclerosis. We show that expression of IL-10 in the spinal axis of SOD1-G93A mice altered the immune milieu and significantly prolonged survival. These data establish an efficient paradigm for somatic transgene delivery of therapeutic biologics to the spinal cord of mice.

With the pace of scientific advancement accelerating rapidly, new methods are needed for experimental neuroscience to quickly and easily manipulate gene expression in the mouse brain. Here we describe a technique first introduced by Passini and Wolfe for direct intracranial delivery of virally-encoded transgenes into the neonatal mouse brain. In its most basic form, the procedure requires only an ice bucket and a microliter syringe. However, the protocol can also be adapted for use with stereotaxic frames to improve consistency for researchers new to the technique. The method relies on the ability of adeno-associated virus (AAV) to move freely from the cerebral ventricles into the brain parenchyma while the ependymal lining is still immature during the first 12-24 hr after birth. Intraventricular injection of AAV at this age results in widespread transduction of neurons throughout the brain. Expression begins within days of injection and persists for the lifetime of the animal. Viral titer can be adjusted to control the density of transduced neurons, while co-expression of a fluorescent protein provides a vital label of transduced cells. With the rising availability of viral core facilities to provide both off-the-shelf, pre-packaged reagents and custom viral preparation, this approach offers a timely method for manipulating gene expression in the mouse brain that is fast, easy, and far less expensive than traditional germline engineering.

The neonatal intraventricular injection of adeno-associated virus has been shown to transduce neurons widely throughout the brain, but its full potential for experimental neuroscience has not been adequately explored. We report a detailed analysis of the method's versatility with an emphasis on experimental applications where tools for genetic manipulation are currently lacking. Viral injection into the neonatal mouse brain is fast, easy, and accesses regions of the brain including the cerebellum and brainstem that have been difficult to target with other techniques such as electroporation. We show that viral transduction produces an inherently mosaic expression pattern that can be exploited by varying the titer to transduce isolated neurons or densely-packed populations. We demonstrate that the expression of virally-encoded proteins is active much sooner than previously believed, allowing genetic perturbation during critical periods of neuronal plasticity, but is also long-lasting and stable, allowing chronic studies of aging. We harness these features to visualise and manipulate neurons in the hindbrain that have been recalcitrant to approaches commonly applied in the cortex. We show that viral labeling aids the analysis of postnatal dendritic maturation in cerebellar Purkinje neurons by allowing individual cells to be readily distinguished, and then demonstrate that the same sparse labeling allows live in vivo imaging of mature Purkinje neurons at a resolution sufficient for complete analytical reconstruction. Given the rising availability of viral constructs, packaging services, and genetically modified animals, these techniques should facilitate a wide range of experiments into brain development, function, and degeneration.

Adeno-associated virus (AAV) mediated gene expression is a powerful tool for gene therapy and preclinical studies. A comprehensive analysis of CNS cell type tropism, expression levels and biodistribution of different capsid serotypes has not yet been undertaken in neonatal rodents. Our previous studies show that intracerebroventricular injection with AAV2/1 on neonatal day P0 results in widespread CNS expression but the biodistribution is limited if injected beyond neonatal day P1. To extend these observations we explored the effect of timing of injection on tropism and biodistribution of six commonly used pseudotyped AAVs delivered in the cerebral ventricles of neonatal mice. We demonstrate that AAV2/8 and 2/9 resulted in the most widespread biodistribution in the brain. Most serotypes showed varying biodistribution depending on the day of injection. Injection on neonatal day P0 resulted in mostly neuronal transduction, whereas administration in later periods of development (24–84 hours postnatal) resulted in more non-neuronal transduction. AAV2/5 showed widespread transduction of astrocytes irrespective of the time of injection. None of the serotypes tested showed any microglial transduction. This study demonstrates that both capsid serotype and timing of injection influence the regional and cell-type distribution of AAV in neonatal rodents, and emphasizes the utility of pseudotyped AAV vectors for translational gene therapy paradigms.

Accumulation and aggregation of amyloid beta peptide 1-42 (Abeta42) in the brain has been hypothesized as triggering a pathological cascade that causes Alzheimer disease (AD). To determine whether selective targeting of Abeta42 versus Abeta40 or total Abeta is an effective way to prevent or treat AD, we compared the effects of passive immunization with an anti-Abeta42 mAb, an anti-Abeta40 mAb, and multiple Abeta(1-16) mAbs. We established in vivo binding selectivity of the anti-Abeta42 and anti-Abeta40 mAbs using novel TgBRI-Abeta mice. We then conducted a prevention study in which the anti-Abeta mAbs were administered to young Tg2576 mice, which have no significant Abeta deposition, and therapeutic studies in which mAbs were administered to Tg2576 or CRND8 mice with modest levels of preexisting Abeta deposits. Anti-Abeta42, anti-Abeta40, and anti-Abeta(1-16) mAbs attenuated plaque deposition in the prevention study. In contrast, anti-Abeta42 and anti-Abeta40 mAbs were less effective in attenuating Abeta deposition in the therapeutic studies and were not effective in clearing diffuse plaques following direct injection into the cortex. These data suggest that selective targeting of Abeta42 or Abeta40 may be an effective strategy to prevent amyloid deposition, but may have limited benefit in a therapeutic setting.

The amyloid beta peptide aggregates into amyloid plaques at presymptomatic stages of Alzheimer's disease, but the temporal relationship between plaque formation and neuronal dysfunction is poorly understood. Here we demonstrate that the connectivity of the peripheral olfactory neural circuit is perturbed in mice overexpressing human APPsw (Swedish mutation) before the onset of plaques. Expression of human APPsw exclusively in olfactory sensory neurons also perturbs connectivity with associated reductions in odour-evoked gene expression and olfactory acuity. By contrast, olfactory sensory neuron axons project correctly in mice overexpressing wild-type human amyloid precursor protein throughout the brain and in mice overexpressing M671V human APP, a missense mutation that reduces amyloid beta production, exclusively in olfactory sensory neurons. Furthermore, expression of Aβ40 or Aβ42 solely in the olfactory epithelium disrupts the olfactory sensory neuron axon targeting. Our data indicate that altering the structural connectivity and function of highly plastic neural circuits is one of the pleiotropic actions of soluble human amyloid beta.

Frontotemporal lobar degeneration (FTLD) has been subdivided based on the main pathology found in the brains of affected individuals. When the primary pathology is aggregated, hyperphosphorylated tau, the pathological diagnosis is FTLD-tau. When the primary pathology is cytoplasmic and/or nuclear aggregates of phosphorylated TAR-DNA-binding protein (TDP-43), the pathological diagnosis is FTLD-TDP. Notably, TDP-43 pathology can also occur in conjunction with a number of neurodegenerative disorders; however, unknown environmental and genetic factors may regulate this TDP-43 pathology. Using transgenic mouse models of several diseases of the central nervous system, we explored whether a primary proteinopathy might secondarily drive TDP-43 proteinopathy. We found abnormal, cytoplasmic accumulation of phosphorylated TDP-43 specifically in two tau transgenic models, but TDP-43 pathology was absent in mouse models of Aβ deposition, α-synucleinopathy or Huntington’s disease. Though tau pathology showed considerable overlap with cytoplasmic, phosphorylated TDP-43, tau pathology generally preceded TDP-43 pathology. Biochemical analysis confirmed the presence of TDP-43 abnormalities in the tau mice, which showed increased levels of high molecular weight, soluble TDP-43 and insoluble full-length and ~35 kD TDP-43. These data demonstrate that the neurodegenerative cascade associated with a primary tauopathy in tau transgenic mice can also promote TDP-43 abnormalities. These findings provide the first in vivo models to understand how TDP-43 pathology may arise as a secondary consequence of a primary proteinopathy. Electronic supplementary material The online version of this article (doi:10.1007/s00401-013-1123-8) contains supplementary material, which is available to authorized users.

Introduction A hallmark of Alzheimer's disease (AD) is the presence of senile plaques composed of aggregated amyloid β (Aβ) peptides. Pathological aging (PA) is a postmortem classification that has been used to describe brains with plaque pathology similar in extent to AD, minimal cortical tau pathology, and no accompanying history of cognitive decline in the brain donor prior to death. PA may represent either a prodromal phase of AD, a benign form of Aβ accumulation, or inherent individual resistance to the toxic effects of Aβ accumulation. To attempt to distinguish between these possibilities we have systematically characterized Aβ peptides in a postmortem series of PA, AD and non-demented control (NDC) brains. Methods Aβ was sequentially extracted with tris buffered saline (TBS), radioimmunoprecipitation buffer (RIPA), 2% sodium dodecyl sulfate (SDS) and 70% formic acid (FA) from the pre-frontal cortex of 16 AD, eight PA, and six NDC patients. These extracts were analyzed by 1) a panel of Aβ sandwich ELISAs, 2) immunoprecipitation followed by mass spectrometry (IP/MS) and 3) western blotting. These studies enabled us to asses Aβ levels and solubility, peptide profiles and oligomeric assemblies. Results In almost all extracts (TBS, RIPA, 2% SDS and 70% FA) the average levels of Aβ1-40, Aβ1-42, Aβ total, and Aβx-42 were greatest in AD. On average, levels were slightly lower in PA, and there was extensive overlap between Aβ levels in individual PA and AD cases. The profiles of Aβ peptides detected using IP/MS techniques also showed extensive similarity between the PA and AD brain extracts. In select AD brain extracts, we detected more amino-terminally truncated Aβ peptides compared to PA patients, but these peptides represented a minor portion of the Aβ observed. No consistent differences in the Aβ assemblies were observed by western blotting in the PA and AD groups. Conclusions We found extensive overlap with only subtle quantitative differences between Aβ levels, peptide profiles, solubility, and SDS-stable oligomeric assemblies in the PA and AD brains. These cross-sectional data indicate that Aβ accumulation in PA and AD is remarkably similar. Such data would be consistent with PA representing a prodromal stage of AD or a resistance to the toxic effects of Aβ.