Seeding properties of amyloid-beta and tau in the cerebrospinal fluid

Alzheimer’s disease (AD) is the most common neurodegenerative disorder, and its prevalence is still increasing. However, currently there are no treatment options available, nor reliable presymptomatic biomarkers for its diagnosis. Neuropathologically, AD is characterized by aggregates (plaques and cerebrovascuar deposits; and tangles) composed of two different proteins: amyloid-β (Aβ) and tau, respectively. Brain derived Aβ and tau exhibit prion-like properties, as based on recent studies. Small and soluble Aβ species have been identified as the most potent Aβ seeds, and they may be present in the CSF which could be of a diagnostic value. Injection of CSF obtained from AD patients into APP23 transgenic mice however did not result into induction or acceleration of amyloidosis, indicating that human CSF Aβ is not seed competent, in contrast to brain Aβ (Fritschi et al. 2014). In order to test this hypothesis further, we injected susceptible mice with APP23 mice’ CSF, containing Aβ at higher amounts than the human CSF, and sacrificed the mice after longer seeding time. As a result, we did not see a significant seeding effect, confirming that CSF Aβ does not show relevant prion-like properties in vivo. Next, we looked at the prion-like properties of tau in the CSF. Since tau concentration in the CSF increases with progression of AD, unlike Aβ levels, it is possible that seeding competent tau species might reach the CSF compartment. For this purpose, we collected CSF from aged, tangle-bearing P301S mice and injected it into young, pretangle stage P301S mice. As a result, we observed significantly higher number of hyperphosphorylated tau inclusions in the injected hippocampus, indicating that CSF tau can induce tau hyperphosphorylation in the host mice. This could be indicative of an early prion-like seeding response. We then investigated whether CSF obtained from human tauopathy patients could induce tau aggregation using the same methodology. The collection of human CSF samples as well as the analysis of human CSF seeded mice is still ongoing. Tau protein fragments have been identified in both the CSF and the brain of AD patients, however it is not clear what is their role in the progression of the disease. Recent studies suggest that tau fragmentation, rather than aggregation per se, may play an important role in neurodegeneration. To study the relation between truncated and full-length tau in vivo and shed more light on this question, we used an inducible mouse line expressing truncated 3R tau, and crossed it with full-length tau-expressing mice (3R, or 4R with and without a mutation). As a result, mice exhibited severe neuronal loss and motor palsy in the absence of tau aggregation. However, they recovered once the expression of fragmented tau was ceased, except for the 3R-expressing mice. This shows the importance of fragmented tau for toxicity and points at new therapeutic targets in the treatment of tauopathies. Taken together, the results presented here point at the possible use of CSF tau in the development of future AD diagnostic essays, and implicate tau truncation as a potential pharmacological target in tauopathies

Monitoring mouse brain perfusion with hybrid magnetic resonance optoacoustic tomography

Progress in brain research critically depends on the development of next-generation multi-modal imaging tools capable of capturing transient functional events and multiplexed contrasts noninvasively and concurrently, thus enabling a holistic view of dynamic events in vivo. Here we report on a hybrid magnetic resonance and optoacoustic tomography (MROT) system for murine brain imaging, which incorporates an MR-compatible spherical matrix array transducer and fiber-based light illumination into a 9.4 T small animal scanner. An optimized radiofrequency coil has further been devised for whole-brain interrogation. System's utility is showcased by acquiring complementary angiographic and soft tissue anatomical contrast along with simultaneous dual-modality visualization of contrast agent dynamics in vivo

Hybrid fiber optic-fMRI for multimodal cell-specific recording and manipulation of neural activity in rodents

Significance: Multiscale imaging holds particular relevance to neuroscience, where it helps integrate the cellular and molecular biological scale, which is most accessible to interventions, with holistic organ-level evaluations, most relevant with respect to function. Being inextricably interdisciplinary, multiscale imaging benefits substantially from incremental technology adoption, and a detailed overview of the state-of-the-art is vital to an informed application of imaging methods. Aim: In this article, we lay out the background and methodological aspects of multimodal approaches combining functional magnetic resonance imaging (fMRI) with simultaneous optical measurement or stimulation. Approach: We focus on optical techniques as these allow, in conjunction with genetically encoded proteins (e.g. calcium indicators or optical signal transducers), unprecedented read-out and control specificity for individual cell-types during fMRI experiments, while leveraging non-interfering modalities. Results: A variety of different solutions for optical/fMRI methods has been reported ranging from bulk fluorescence recordings via fiber photometry to high resolution microscopy. In particular, the plethora of optogenetic tools has enabled the transformation of stimulus-evoked fMRI into a cell biological interrogation method. We discuss the capabilities and limitations of these genetically encoded molecular tools in the study of brain phenomena of great methodological and neuropsychiatric interest—such as neurovascular coupling (NVC) and neuronal network mapping. We provide a methodological description of this interdisciplinary field of study, and focus in particular on the limitations of the widely used blood oxygen level dependent (BOLD) signal and how multimodal readouts can shed light on the contributions arising from neurons, astrocytes, or the vasculature. Conclusion: We conclude that information from multiple signaling pathways must be incorporated in future forward models of the BOLD response to prevent erroneous conclusions when using fMRI as a surrogate measure for neural activity. Further, we highlight the potential of direct neuronal stimulation via genetically defined brain networks towards advancing neurophysiological understanding and better estimating effective connectivity

Cerebrospinal fluid from Alzheimer's disease patients promotes tau aggregation in transgenic mice

Tau is a microtubule stabilizing protein that forms aggregates in Alzheimer's disease (AD). Tau derived from AD patients' brains induces tau aggregation in a prion-like manner when injected into susceptible mouse models.Here we investigated whether cerebrospinal fluid (CSF) collected from patients diagnosed with probable AD or mild cognitive impairment (MCI) likely due to AD harbors a prion-like tau seeding potential. CSF was injected intrahippocampally into young P301S tau transgenic mice. CSF obtained from AD or MCI patients increased hippocampal tau hyperphosphorylation and tau tangle formation in these mice at 4 months post-seeding. Tau pathology was also accentuated in the contralateral hippocampus, and in anterior and posterior directions, indicative of spreading.We provide first evidence for in vivo prion-like properties of AD patients' CSF, accelerating tau pathology in susceptible tau transgenic mice. This demonstrates that biologically active tau seeds reach the CSF compartment in AD. Further studies may help to evaluate strain specific properties of CSF derived tau bioseeds, and to assess their diagnostic potential. What do you want to do ? New mail Cop

Co-expression of truncated and full-length tau induces severe neurotoxicity.

Hyperphosphorylated and aggregated tau protein constitutes the main pathological hallmark of tauopathies, including Alzheimer’s disease (AD). While proteolytically cleaved peptides play a central role in various neurodegenerative disorders, the importance of tau fragmentation for the pathogenesis of tauopathies is still being debated. Here we studied the toxicity of a human 3 repeat tau151-421 fragment (tau) in vivo, using a novel inducible transgenic mouse model (TAU62 mice). These mice developed a slowly progressive motor phenotype and histopathological lesions compatible with axonal damage. To mimic the disease situation, where tau fragmentation occurs in presence of full-length tau, we co-expressed tau with human full-length human P301S tau (P301SxTAU62 mice). This resulted in severe neuronal dysfunction with rapidly progressive motor palsy and a wide spectrum of tauopathy associated changes, notably in the absence of tau tangles or filaments. Strikingly, these changes were widely reversible when tau expression was halted. In contrast, co-expression of full-length human 3 repeat tau with P301S tau (P301SxALZ31 mice) did not result in a drastic phenotype. We conclude that tau fragments can potentiate full-length tau toxicity in vivo and in this way may contribute to the pathogenesis of tauopathies. The fact that this happened in the absence of tangle formation may reflect an early phase of tauopathies, in which functional changes induced by yet soluble toxic tau species can still be partly reversible by curative treatments

Levels of sarkosyl extracted insoluble tau were significantly reduced in the forebrain of P301S mice after 5 months of long-term rapamycin treatment (R, n = 6) when compared to vehicle treated mice (V, n = 5) (A, 5MT group; Western blot using BR134 antibody).

<p>A comparable lowering of insoluble tau was obtained by late short-term rapamycin administration over 6 weeks (B; 6WT group, n = 6/6). In parallel, the accumulation of tau hyperphosphorylated at the AT8 and AT100 epitopes was significantly lowered (C; 6WT group, n = 6/6). Quantification of tau Western blots was subjected to unpaired T-tests and T-tests adjusted for unequal variances (Welch-Test), yielding both similar results. *<i>p<0.05 and</i> **<i>p<0.01.</i></p

The extensive cortical tau tangle pathology present in 5.5 months old vehicle (veh) treated P301S mice (A) was widely attenuated in long-term rapamycin (rapa) treated mice (B).

<p>The lowering in tangle formation was most pronounced in the motor cortex (C: left vehicle treated/right rapamycin) and associated with reduced pathological tau hyperphosphorylation at the AT8 and AT100 epitopes (D, E). In parallel, cortical astrogliosis was diminished following rapamycin treatment (F). While there was a trend towards a reduction of the sparse tangles in the hippocampus, the advanced tau pathology in the brain stem however was not significantly ameliorated by rapamycin (G, H). Short-term treatment at 3 months of age for 6 weeks again resulted in a marked reduction of cortical tangles (I) (A–H: 5MT group; I: 6WT. A–C, G–I: Gallyas silver stain; D: AT8 IHC; E: AT100 IHC; F: GFAP IHC. Bar in A equals 300 µm in A and B, 150 µm in C, G and I, 75 µm in D–F, 600 µm in H).</p

Consistent with cerebral mTOR inhibition, phosphorylation of S6 (S6P) was significantly reduced following rapamycin administration (A, 5MT group, n = 4/4).

<p>Compatible with an induced autophagy pathway, LC3II levels were increased upon rapamycin treatment (B, 6WT group, n = 6/6). High levels of the autophagy associated proteins p62 and LC3 were measured in aged vehicle treated P301S transgenic mice (C; 5MT Vehicle, n = 4). This accumulation of p62 and LC3 was prevented by long-term rapamycin administration, pointing towards a restored autophagic flux (C; 5MT Rapa, n = 4). Forebrain tissue was used and data was analyzed by ANOVA. *<i>p<0.05,</i> **<i>p<0.01, and</i> ***<i>p<0.001.</i></p

After long-term rapamycin treatment (n = 6), unbiased stereology confirmed a significant reduction of cortical tau tangles to only 14% of the amount of tangles seen in vehicle treated (n = 5) P301S mice (vehicle = 100%).

<p>The number of AT8 stained cells containing hyperphosphorylated tau was reduced to 30% in aged rapamycin treated mice compared to controls (100%) (5MT group). A significant attenuation of Gallyas-stained (to 39% of controls) and AT8-positive cells (to 46% of controls) was also achieved by late short-term rapamycin treatment (6WT, n = 6/6). The reduction of tangles in the hippocampus (to 33% of controls) and the brain stem (to 72% of controls) did not reach the level of significance adjusted for multiple testing. Pairwise reduction of Gallyas or AT8 positive counts was analysed using one-sample T-tests per brain region. Significant p-values, adjusted for the multiple comparisons of all 6 tested groups by the Holm-Bonferroni method, are outlined as follows: *<i>p<0.05,</i> **<i>p<0.01, and</i> ***<i>p<0.001.</i></p