Fig 1.
Chronogram for evaluating rapamycin in tau-mediated neurotoxicity for the perforant pathway, and biochemical evidence for target engagement in the mouse brain.
Schematic of the time course for systemic treatment with rapamycin or vehicle after AAV-hTauP301L microinjections, in relation to the neuropathological assessments of the lateral entorhinal cortex (LEC) neurons and lateral perforant pathway synapses. (B) Western blot analysis of phosphorylation of the mTOR substrate P70S6 kinase on pThr389 and proteolytic generation of the autophagic vacuole membrane protein LC3 in hippocampus 3 weeks after viral vector delivery and drug treatment. Actin served as a loading control. (C) Quantitative analysis of P70S6 kinase phosphorylation and LC3 cleavage. Compared to hippocampus of vehicle-treated controls (V), chronic rapamycin (R) significantly attenuated mTOR substrate phosphorylation (*p = 0.013) and increased autophagic flux as monitored by cleaved LC3 (-II) relative to the full-length form (LC3-I; **p<0.01). Comparable results were obtained from the neocortex.
Fig 2.
Protective effect of rapamycin for perforant pathway neurons of origin in layer II of the lateral entorhinal cortex.
(A,D,G)–Lateral entorhinal cortex, contralateral hemisphere, vehicle treatment. (B,E,H)–Lateral entorhinal cortex ipsilateral to human tau gene delivery, vehicle treatment. (C,F,I)—Lateral entorhinal cortex, ipsilateral to human tau gene delivery, rapamycin treatment. (A-C) Immunohistochemical localization of pTau202/205 using the AT8 monoclonal antibody. Note that ipsilateral but not contralateral to the viral vector-based human tau gene delivery, pTau202/205 localizes preferentially in layer II neuronal perikarya and their proximal dendritic processes. Chronic rapamycin does not appreciably alter total pTau202/205 levels. (D-F) Immunohistochemical staining for NeuN-positive surviving lateral entorhinal neurons. Layer II of the lateral entorhinal cortex is denoted by the borders shown in D. Note the marked loss of layer II neurons after expression of pathological human tau (compare E with the contralateral hemisphere in D) and attenuation of the NeuN-positive neuronal loss by rapamycin treatment (F). (G-H) Analysis of neuronal survival and non-neuronal proliferation with cresyl violet staining. Compared to the control lateral entorhinal cortex (G), in entorhinal layer II ipsilateral to pathological human tau expression most of the healthy, plump, lightly-stained neuronal perikarya disappear, and are replaced by small, dark non-neuronal cells (H). Rapamycin preserves many of the healthy neurons and partially suppresses the non-neuronal cell proliferation (I). Scale bar = 75 μm.
Table 1.
Systemic rapamycin attenuates pathological human tau-induced perforant pathway neuronal degeneration, synapse loss, trans-synaptic tau transfer, and reactive gliosis without influencing transgene expression.
Fig 3.
Dose-dependent tau neurotoxicity for lateral perforant pathway neurons is tied to trans-synaptic expansion of human tau expression, and both are reduced by rapamycin.
Left column–NeuN immunohistochemistry in the lateral entorhinal cortex 5 weeks after AAV-based human tau gene delivery. Right column–Total human tau immunohistochemistry in the ipsilateral dentate gyrus. A) After delivery of 0.5 billion particles of AAV-hTauP301L (low dose), there is no appreciable neuronal loss in the lateral entorhinal layer II (see also Siman et al., 2013). In the dentate gyrus under these conditions, human tau is confined to the terminal field for the lateral perforant pathway in the outer molecular layer (OML). Additional abbreviations: MML- middle molecular layer; IML- inner molecular layer; GCL- granule cell layer. B) After delivery of 1.5 billion particles of AAV-hTauP301L (high dose), there is extensive loss of lateral entorhinal layer II neurons. In the dentate gyrus, human tau is present not only in the lateral perforant pathway terminal field in the OML, but also in scattered granule neurons in the GCL. C) Chronic treatment with vehicle does not appreciably alter the toxicity of pathological human tau for the layer II neurons of lateral entorhinal cortex or the spread of human tau expression to some of the dentate granule neurons. D) Chronic rapamycin treatment partially protects the perforant pathway layer II neurons from tau-mediated degeneration, and also partially inhibits the trans-synaptic spread of human tau expression to their granule neuron targets. Scale bar (left) = 200 μm; Scale bar (right) = 40 μm (A,B), 20 μm (C,D).
Fig 4.
Co-delivery of AAV-eGFP with a toxic dose of AAV-hTauP301L to the lateral entorhinal cortex leads to trans-synaptic expression of eGFP as well as human tau in dentate granule neurons.
A) After intra-entorhinal delivery of the eGFP vector alone, eGFP is expressed in the lateral perforant pathway projection as it traverses the stratum lacunosum-moleculare (SLM) of the hippocampal CA1 sector, before perforating the hippocampal fissure to form synaptic connections on dentate granule neurons in the outer molecular layer (OML). Note that the middle molecular layer (MML), inner molecular layer (IML), granule cell layer (GCL), and hilus (HIL) are devoid of eGFP. B) In contrast, when the eGFP vector is co-delivered to lateral entorhinal cortex along with a toxic dose of the pathological human tau vector, eGFP expression mimics human tau expression by expanding to scattered dentate granule neurons (arrowheads). C) Under high magnification image deconvolution microscopy, the eGFP-expressing cells in the granule cell layer are confirmed as dentate granule neurons based on their morphology. Scale bar = 150 μm (A,B), 15 μm (C).
Fig 5.
Rapamycin protects against tau-mediated lateral perforant pathway synapse loss.
Timm staining in the dentate gyrus demarcates the afferent lamination of presynaptic terminal zinc. Left four panels, vehicle treatment: Contralateral to AAV vector injection (far left) in both blades of the dentate gyrus, zinc staining reveals a relatively dark zone in the outer molecular layer (OML) corresponding to the lateral entorhinal cortex and perforant pathway afferents. Neighboring afferent laminae in the stratum lacunosum-moleculare (SLM) above the hippocampal fissure (HF) for the suprapyramidal blade or the thalamus (THAL) for the infrapyramidal blade, or the middle molecular layer (MML) are demarcated by their lower synaptic zinc content. At 5 weeks after unilateral expression of pathological human tau in the lateral perforant pathway and treatment with vehicle, the lateral perforant pathway terminal field in the OML has largely degenerated in both blades of the ipsilateral dentate gyrus, concomitant with an expansion of the relatively zinc-poor medial perforant pathway afferents terminating in the MML. Right four panels, rapamycin treatment: Chronic rapamycin spares the relatively zinc-rich lateral perforant pathway synapses in the OML of both blades of the dentate gyrus. Scale bar = 40 μm.
Fig 6.
Rapamycin reduces pathological tau-triggered reactive microgliosis in the lateral perforant pathway synaptic field.
Microglial activation triggered by pathological tau-induced neurodegeneration were distinguished from basal microglia based on their marked increase in immunohistochemical labeling for CR3/CD11b. (A) Cells with modest CR3 expression and the morphology of microglia (inset) were dispersed throughout the hippocampal dentate gyrus contralateral to viral vector delivery. (B) At 3 weeks after expression of pathological human tau in the lateral perforant pathway in mice treated with vehicle, dense bands of microglia with increased CR3 expression and enlarged processes were observed in the lateral perforant pathway terminal field in the outer molecular layer of both blades of the dentate gyrus. Whereas chronic rapamycin treatment did not appreciably alter basal CR3 expression in the contralateral hemisphere (C), it markedly attenuated the CR3 induction in reactive microglia in the lateral perforant pathway synaptic field (D). Abbreviations: HF- hippocampal fissure; OML- dentate outer molecular layer; MML- dentate middle molecular layer; IML- dentate inner molecular layer; GCL-dentate granule cell layer. Scale bar = 100 μm.
Fig 7.
Rapamycin inhibits pathological tau-activated innate immunity in the lateral perforant pathway synaptic field.
Microglial-mediated innate immunity in response to pathological tau-induced neurodegeneration was evaluated by immunohistochemical labeling for mouse IgG. (A) Cells with modest IgG expression were dispersed throughout the hippocampal dentate gyrus contralateral to viral vector delivery. (B) At 3 weeks after expressing pathological human tau in the lateral perforant pathway in mice treated with vehicle, a dense band of microglia with increased IgG expression was observed in the dentate gyrus outer molecular layer. (C) The morphology of IgG-expressing cells confirmed their identification as microglia. Whereas chronic rapamycin treatment modestly reduced microglial IgG expression in the contralateral hemisphere (D), it markedly attenuated the reactive microgliosis-associated increase in IgG expression in the lateral perforant pathway synaptic field (E, low magnification; F, high magnification). Essentially identical findings were made in the infrapyramidal blade of the dentate gyrus. Scale bar = 20 μm (A,B,D,E), 10 μm (C,F).
Fig 8.
Rapamycin attenuates pathological tau-induced reactive astrogliosis in the lateral entorhinal cortex.
Astroglial activation in response to pathological tau-induced neurodegeneration was evaluated by immunohistochemical staining for GFAP. (A,C) In lateral entorhinal cortex contralateral to viral vector-based gene delivery, astrocytes express relatively modest levels of GFAP (A, low magnification) and are small in size (C, high magnification). Ipsilateral to viral vector-based expression of pathological human tau and chronic vehicle treatment, the neuronal loss in layer II is accompanied by reactive astrogliosis, characterized by increased GFAP expression (B) and glial hypertrophy (D). In contrast, chronic rapamycin treatment markedly reduced the reactive astrogliosis in the pathological human tau-expressing lateral entorhinal cortex (compare E to B). Scale bar = 100 μm (A,B,E); 20 μm (C,D).