Behavioral and neuropathological characterization over the adult lifespan of the human tau knock-in mouse

Tau is a microtubule-associated protein with a diverse functional repertoire linked to neurodegenerative disease. Recently, a human tau knock-in (MAPT KI) mouse was developed that may overcome many limitations associated with current animal models used to study tau. In MAPT KI mice, the entire murine Mapt gene was replaced with the human MAPT gene under control of the endogenous Mapt promoter. This model represents an ideal in vivo platform to study the function and dysfunction of human tau protein. Accordingly, a detailed understanding of the effects MAPT KI has on structure and function of the CNS is warranted. Here, we provide a detailed behavioral and neuropathological assessment of MAPT KI mice. We compared MAPT KI to wild-type (WT) C57BL/6j mice in behavioral assessments of anxiety, attention, working memory, spatial memory, and motor performance from 6 to 24 months (m) of age. Using immunohistological and biochemical assays, we quantified markers of glia (microglia, astrocytes and oligodendrocytes), synaptic integrity, neuronal integrity and the cytoskeleton. Finally, we quantified levels of total tau, tau isoforms, tau phosphorylation, and tau conformations. MAPT KI mice show normal cognitive and locomotor behavior at all ages, and resilience to mild age-associated locomotor deficits observed in WT mice. Markers of neuronal and synaptic integrity are unchanged in MAPT KI mice with advancing age. Glial markers are largely unchanged in MAPT KI mice, but glial fibrillary acidic protein is increased in the hippocampus of WT and MAPT KI mice at 24 m. MAPT KI mice express all 6 human tau isoforms and levels of tau remain stable throughout adulthood. Hippocampal tau in MAPT KI and WT mice is phosphorylated at serine 396/404 (PHF1) and murine tau in WT animals displays more PHF1 phosphorylation at 6 and 12 m. Lastly, we extended previous reports showing that MAPT KI mice do not display overt pathology. No evidence of other tau phosphorylation residues (AT8, pS422) or abnormal conformations (TNT2 or TOC1) associated with pathogenic tau were detected. The lack of overt pathological changes in MAPT KI mice make this an ideal platform for future investigations into the function and dysfunction of tau protein in vivo

Novel Nonphosphorylated Serine 9/21 GSK3β/a Antibodies: Expanding the Toolkit for Studying GSK3 Regulation

Glycogen synthase kinase 3 (GSK3) and are serine/threonine kinases involved in many biological processes. A primary mechanism of GSK3 activity regulation is phosphorylation of N-terminal serine (S) residues (S9 in GSK3b, S21 in GSK3a). Phosphorylation is inhibitory to GSK3 kinase activity because the phosphorylated N-terminus acts as a competitive inhibitor for primed substrates. Despite widespread interest in GSK3 across most fields of biology, the research community does not have reagents that specifically react with nonphosphoS9/21 GSK3a/b (the so-called active form). Here, we describe two novel monoclonal antibodies that specifically react with nonphosphoS9/21 GSK3a/b in multiple species (human, mouse and rat). One of the antibodies is specific for nonphospho-S9 GSK3b (clone 12B2) and one for nonphospho-S9/21 GSK3a/b (clone 15C2). These reagents were validated for specificity and reactivity in several biochemical and immunochemical assays, and they show linear detection of nonphosphoS GSK3. Finally, these reagents provide significant advantages in studying GSK3 regulation. We used 12B2 (due to its specificity for GSK3bs) to study the regulation of S9 phosphorylation by Akt and protein phosphatases, and to demonstrate that protein phosphatase inhibition reduces nonphospho-S9 GSK3b levels and lowers kinase activity within cells. The ability to use the same reagent across biochemical, immunohistological and kinase activity assays provides a powerful approach for studying serine-dependent regulation of GSK3a/b

A Method for Combining RNAscope In Situ Hybridization with Immunohistochemistry in Thick Free-Floating Brain Sections and Primary Neuronal Cultures

<div><p>In situ hybridization (ISH) is an extremely useful tool for localizing gene expression and changes in expression to specific cell populations in tissue samples across numerous research fields. Typically, a research group will put forth significant effort to design, generate, validate and then utilize <i>in situ</i> probes in thin or ultrathin paraffin embedded tissue sections. While combining ISH and IHC is an established technique, the combination of RNAscope ISH, a commercially available ISH assay with single transcript sensitivity, and IHC in thick free-floating tissue sections has not been described. Here, we provide a protocol that combines RNAscope ISH with IHC in thick free-floating tissue sections from the brain and allows simultaneous co-localization of genes and proteins in individual cells. This approach works well with a number of ISH probes (e.g. small proline-rich repeat 1a, βIII-tubulin, tau, and β-actin) and IHC antibody stains (e.g. tyrosine hydroxylase, βIII-tubulin, NeuN, and glial fibrillary acidic protein) in rat brain sections. In addition, we provide examples of combining ISH-IHC dual staining in primary neuron cultures and double-ISH labeling in thick free-floating tissue sections from the brain. Finally, we highlight the ability of RNAscope to detect ectopic DNA in neurons transduced with viral vectors. RNAscope ISH is a commercially available technology that utilizes a branched or “tree” <i>in situ</i> method to obtain ultrasensitive, single transcript detection. Immunohistochemistry is a tried and true method for identifying specific protein in cell populations. The combination of a sensitive and versatile oligonucleotide detection method with an established and versatile protein assay is a significant advancement in studies using free-floating tissue sections.</p></div

Control sections for ISH-IHC dual labeling in thick free-floating sections.

<p>A) Tissues were labeled with a rat positive control probe (i.e. rat peptidylprolyl isomerase B gene) provided by ACD. Clear ISH signal and IHC staining were observed. This probe acts as a positive control because it is present ubiquitously throughout rat tissue, albeit at modest levels in the cells depicted. B) Tissues were labeled with a negative control probe (i.e. <i>Bacillus subtilis</i> dihydrodipicolinate reductase gene) provided by ACD. Very infrequent ISH puncta are present, while strong IHC is clear. This probe was used as a negative control because it is a bacterial transcript not present in rat tissue. C) Tissue was labeled with Sprr1a ISH, but the primary TH antibody was excluded. As expected, strong Sprr1a ISH is apparent in the SN of the lesioned hemisphere (i.e. brown ISH puncta), but no TH IHC is present (i.e. lack of blue staining). D) To ensure the signals were dependent upon the Sprr1a ISH probe and TH primary antibody we omitted the Sprr1a probe and the TH primary antibody. As expected, this results in no signal for ISH (i.e. no brown puncta) or IHC (i.e. no blue staining). Scale bar: A-D = 50μm.</p

RNAscope ISH combined with IHC in thick free-floating rat brain tissue sections.

<p>The dopaminergic nigrostriatal system of rats was unilaterally lesioned using 6-OHDA (a dopaminergic neurotoxin) delivered to the striatum. One week later the brains were collected and tissue was processed for Sprr1a ISH (brown) and tyrosine hydroxylase (TH) IHC (blue). A) Low magnification images clearly depict the loss of dopaminergic (i.e. TH+) neurons in the lesioned (right side) substantia nigra compared to the unlesioned hemisphere (left side). Note the presence of substantial Sprr1a ISH signal in the lesioned hemisphere, and the lack of signal in the unlesioned hemisphere. B and C) High magnification images of a 20μm thick section show clear dual labeling with ISH and IHC in the lesioned side (C), but not the unlesioned side (B). D and E) Images from a 40μm thick section show similar results. F-H) Neurons in the lesioned hemisphere exhibited little to no ISH with strong IHC (F), intermediate ISH and IHC (G), and strong ISH with little to no IHC (H). Scale bars: A = 500μm, B-E = 40μm and F-H = 20μm.</p

Illustration of RNAscope branched ISH method.

<p>RNAscope utilizes a branched ISH method that provides significant amplification and discrete detection of single transcripts. (A) RNAscope ISH produces puncta of signal that represent a single mRNA transcript. It is noteworthy, that at a certain level the amount of transcript can be elevated to a level where individual puncta are difficult to discern within single cells (see Figs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120120#pone.0120120.g002" target="_blank">2I</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120120#pone.0120120.g005" target="_blank">5D</a> as examples). (B) This level of sensitivity is achieved by initially hybridizing multiple tandem “Z” probes to the target transcript (these primers typically target 50bp long sequences and multiple sets of primers are made against the target molecule, ACD offers custom designed primers). Then a pre-amplifier probe is bound to the tandem Z probe sets, followed by the addition of multiple amplifier probes to the pre-amplifier probe. Finally, a number of detection probes that are conjugated to horseradish peroxidase (HRP) enzyme are added onto the amplifier probes and HRP substrate is added (i.e. diaminobenzidine, DAB). The resulting conversion of DAB to a brown colored reaction product by all of the HRPs bound the branched ISH complex produces discrete puncta. Alternatively, alkaline phosphatase (two-plex RNAscope) or fluorophores (multi-plex RNAscope) are conjugated to the detection probes for the appropriate version of the assay.</p

The versatility of ISH-IHC dual staining method.

<p>A number of combinations with different ISH and IHC stains were performed. A) Rat βIII-tubulin ISH (brown) and βIII-tubulin IHC (blue) were combined to label the mRNA and protein of the same target. B) Rat βIII-tubulin ISH was combined with IHC for NeuN, a common neuron-specific marker. C) Rat β-actin ISH was combined with IHC for GFAP, a common astrocyte-specific marker. Both B and C confirm that cell type-specific markers are compatible with this ISH-IHC dual staining method. D) Sprr1a ISH was combined with GFAP IHC to demonstrate that the same ISH marker can be combined with multiple IHC markers (i.e. TH in Figs. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120120#pone.0120120.g002" target="_blank">2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120120#pone.0120120.g005" target="_blank">5</a> and GFAP here). All images were taken in the area of the retrosplenial cortex. Scale bar: A-D = 50μm.</p

Semi-quantitative single-cell analysis of ISH signal in dual labeled (ISH-IHC) thick tissue sections.

<p>A-D) Four groups of TH+ neurons were found with varying amounts of ISH signal in the lesioned hemisphere (using same threshold settings as in <b><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0120120#pone.0120120.g003" target="_blank">Fig. 3</a></b>). Individual neurons contained no/very little (A and E), low (B and F), moderate (C and G), and high (D and H) levels of ISH signal. The red areas in E-H are the pixels analyzed after the thresholds were set. I-L) These groups were distinguishable using the mean object number (I), mean area (J), size of the objects (K) and the fraction of the total area (L). The object number measurement detected significant differences between all four groups (p<0.05), while all other measurements identified differences between the low, moderate and high level of ISH groups. Scale bar: A-H = 20μm.</p

RNAscope ISH-IHC in primary neuron cultures, dual RNAscope ISH detection, and detection of ectopic viral vector DNA.

<p>A) Primary rat neurons were processed for tau ISH and βIII-tubulin IHC. Clear puncta are present for the tau ISH signal (red), while neuronal cell bodies and processes are clearly labeled with tubulin IHC (blue). B-E) Color thresholding (using the YUV index) allows clear separation of ISH signal (red puncta) from the IHC signal (blue staining) in primary neurons. Here, the YUV index was used and Y was 0–135, U was 0–255 and V was 135–255. The pixels within the threshold limits are shown by the bright red overlay. F) Dual ISH for tau (red) and βIII-tubulin (blue/green) was performed on 40μm thick rat brain sections (cortex depicted). G-J) Rats were unilaterally injected in the striatum with recombinant adeno-associated viruses (rAAV, serotype 2/5). RNAscope was used to detect rAAV DNA. Robust rAAV DNA ISH signal is detected in the injected striatum (G) compared to the uninjected hemisphere (I). ISH signal was also detected in the SN ipsilateral to the injected striatum because rAAV particles are retrogradely transported to the SN (H), no signal is detected in the contralateral SN (J). Scale bars: A = 50μm, C and E = 20μm, F = 25μm, and J = 50μm.</p