Excessive local host-graft connectivity in aging and amyloid-loaded brain

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Excessive local host-graft connectivity in aging and amyloid-loaded brain

n Judith Thomas1,2,3 , Maria Fernanda Martinez-Reza1,2,3 , Manja Thorwirth1,2 , Yvette Zarb1,2 , Karl-Klaus Conzelmann4 , Stefanie M. Hauck5 *, Sofia Grade1,2 *†‡, Magdalena Götz1,2,6 *‡§ 

 

Transplantation is a clinically relevant approach for brain repair, but much remains to be understood about influences of the disease environment on transplant connectivity. To explore the effect of amyloid pathology in Alzheimer’s disease (AD) and aging, we examined graft connectivity using monosynaptic rabies virus tracing in APP/PS1 mice and in 16- to 18-month-old wild-type (WT) mice. Transplanted neurons differentiated within 4 weeks and integrated well into the host visual cortex, receiving input from the appropriate brain regions for this area. Unexpectedly, we found a prominent several-fold increase in local inputs, in both amyloid-loaded and aged environments. State-of-the-art deep proteome analysis using mass spectrometry highlights complement system activation as a common denominator of environments promoting excessive local input connectivity. These data therefore reveal the key role of the host pathology in shaping the input connectome, calling for caution in extrapolating results from one pathological condition to another. INTRODUCTION There is an urgent medical need for replacement of degenerated neurons after brain injury or neurodegenerative disease (1, 2). Transplantation of fetal or pluripotent stem cell (PSC)–derived neurons is at the forefront of this approach and has successfully achieved clinical improvements (1–8). The easier access and scalability of neurons from induced PSCs for transplantation have further boosted the attempts for clinical translation, e.g., in Parkinson’s disease (PD) patients (9–11). However, rather little is known about neuronal graft integration in other neurodegenerative diseases or physiological aging. To repair neural circuit structure and function, transplanted neurons must connect properly. Aberrant wiring may perturb adequate circuit function, especially in diseases affecting the cerebral cortex, given its high excitatory drive. While the output connectivity of neuronal transplants was found to be remarkably specific many years ago (12–17), the brain-wide analysis of input connectivity and in vivo observation of neuronal activity evoked by stimulation of those afferents or sensory stimulation have been probed more recently (18–22). Fetal cells grafted into the cerebral cortex after neuronal ablation receive brain-wide inputs from the host, as revealed by rabies virus (RABV)–based retrograde monosynaptic tracing (18). These inputs closely resemble the input connectome of the lost cortical neurons (18). Co-registration of RABV-based tracing and three-dimensional (3D) magnetic resonance imaging demonstrated synaptic integration of human neural transplants grafted into different brain regions in mice (23). Similar results have been obtained with cells grafted in a stroke model (20, 22), in a traumatic brain injury (TBI) model (24), or in PD models (21, 25, 26), although the brain-wide input connectome was not quantified. Contrary to acute injuries, characterized by two distinct and in some ways counteracting phases, a proinflammatory acute and an anti-inflammatory chronic phase, conditions differ profoundly during aging and in many neurodegenerative diseases, with progressive and slow synapse and neuron loss and steady changes of the environment (27–29). Reactive gliosis after acute and invasive brain damage, such as stroke and TBI (30, 31), differs profoundly from that in the aging brain or in Alzheimer’s disease (AD) models (32–35). Aging or amyloid-loaded AD brains present vastly different host environments, where transplants survive, but their connectivity remains unknown (36–39). Beyond the lack of connectivity analysis of transplants in these highly relevant conditions, basic principles for new neuron integration into a preexisting circuitry are not known. Transplantation into the adult brain has mostly been performed in models with neuronal loss, but little is known if loss of neurons is a prerequisite to integrate new ones. Synaptic loss is often the first step before neurodegeneration, and it is not known if and how it may influence the integration of new neurons into preexisting but degenerating circuits. In addition, the contribution of reactive gliosis and inflammation to host-graft connectivity has been hardly explored. The influence of inflammation is difficult to deduce from previous studies of human cell transplants, as these xenografts require immunosuppression (20, 22, 36, 40). To disentangle the influence of these parameters on transplant connectivity, we explored aging and amyloid-loaded brain environments in murine models without loss of neurons but with loss of synapses and a different extent of reactive gliosis (33, 35, 41). The aging brain is characterized by gradual cellular and molecular changes like oxidative damage and mitochondrial dysfunction, accumulation of aggregated proteins, and mild inflammation accompanied by mild reactive gliosis (42–44). These age-related changes cause synapse loss, which ultimately leads to impaired function and cognitive decline (42). In addition, aging is the main risk factor for neurodegenerative diseases like AD (45). Hallmarks of AD include the extracellular accumulation of misfolded amyloid- peptide fibrils that aggregate forming plaques, also eliciting astro- and microgliosis and loss of synapses, eventually resulting in network dysfunction and cognitive impairments (46). Thus, it is important to understand how these environments may affect synaptic integration of transplanted neurons to determine the effect of basic parameters, such as synapse loss and reactive gliosis onto graft integration, as well as to illuminate their clinical relevance. RESULTS Characterization of amyloid pathology and aging mouse models To explore the integration of new neurons into an environment with stronger versus weaker pathology and no neuron loss, we chose mouse models of AD and aging, respectively, and used allografts, allowing us to probe these basic principles while simultaneously exploring graft integration into highly relevant dysfunctional environments. First, we compared the extent of reactive gliosis in both models. The APP/PS1 mouse model of AD used here overexpresses human amyloid precursor protein (APP) and presenilin 1 (PS1) in neurons (32) lacking neuronal death in the cerebral cortex (47). These transgenic mice develop early cerebral amyloidosis accompanied by the appearance of hypertrophic microglia and reactive astrocytes and show dendritic spine loss around deposited amyloid plaques (32, 33, 48). Staining for glial fibrillary acidic protein (GFAP) to label reactive astrocytes and ionized calcium binding adaptor molecule 1 (Iba1) to label microglia showed profound reactive gliosis in the cortex of APP/PS1 transgenic mice at 8 months of age compared to age-matched wild-type (WT) control animals of the same background (C57BL/6J) (see Materials and Methods for details; fig. S1, A and B). For a quantitative readout, we assessed the number and morphology of microglia and length of their processes and found significant changes between the control and the APP/PS1 mice. This confirmed high microglial reactivity, with a significant increase in the number of microglia (fig. S1, D, E, and G), as well as of its activation state, with fewer branches and ramifications in transgenic mice compared to WT controls (fig. S1, D, E, and H to J). We then analyzed the glial state in aging cerebral cortices of 16- to 18-month-old WT mice of the same background (C57BL/6J) as the AD model. Both GFAP and Iba1 staining revealed much lower reactivity in cortices of WT aged mice compared to APP/PS1 transgenic mice (fig. S1, B and C). Accordingly, our detailed microglia analysis revealed no significant change in Iba1-stained microglia number or their process length and branching in cortices of 8-month-old versus 16- to 18-month-old WT mice (fig. S1, D, F, and G to J). However, a significantly higher percentage of microglial cells have a large volume covered by their processes in the aged compared to control cortices (fig. S1, K to N). Together, this analysis shows lower levels of reactive gliosis in the aged brain compared to the amyloid pathology, making this comparison very interesting, as both models exhibit altered synaptic dynamics (43, 48, 49). New neurons survive and integrate into cortical circuits in APP/PS1 and aged mice To explore the influence of the distinctive cellular and molecular environments of APP/PS1 and aged brains on synaptic integration of transplanted neurons, we used our previously established paradigm of cell transplantation, circuit mapping, and quantitative connectomics (18). Mouse cortical cells were isolated from embryonic day 14 (E14) C57BL/6J embryos and transduced with a retrovirus encoding the rabies glycoprotein (G), which is required for its retrograde transport, the receptor TVA (avian tumor virus receptor A) allowing for selective infection of these cells by the Env-A-pseudotyped RABV (50), and a fluorescent protein (FP; Fig. 1A). After 3 to 5 days in culture, cells were collected and transplanted into the primary visual cortex (V1) of 8-month-old APP/PS1 transgenic mice. At this age, plaque deposition and reactive gliosis are widespread (fig. S1), as is the decrease in dendritic spines in the absence of neuron loss (32, 47). As controls, APP/PS1 WT littermates of the same background (C57BL/6J) and C57BL/6J mice, both of the same age, were used. As a third condition, we also transplanted cells into 16- to 18-month-old aging C57BL/6J mice (Fig. 1A). In previous work, we had not found any difference in the input connectome to cells cultured and infected in vitro or acutely dissociated from the cortex of E18 Emx1Cre/G-TVA/GFP transgenic mice before transplantation (18). To ensure that this is also the case in the host environments tested here, we also performed a few transplantations with acutely dissociated E18 cortical cells (fig. S2G). Analysis at 5 weeks post-transplantation (wpt) showed that donor cells survived well in all three conditions and no difference in graft size and volume was observed (Fig. 1, B to D, and fig. S2, A to E). To trace the synaptic inputs to transplants developed in all the conditions, we injected EnvA-pseudotyped and G-deleted RABV (50) expressing green fluorescent protein (GFP) or mCherry [referred to as red fluorescent protein (RFP)], depending on donor cell fluorescence. This allowed selective targeting of TVA-expressing donor cells at 4 wpt followed by circuit analysis 1 week thereafter (Fig. 1A). We ensured the reliability of the transsynaptic tracing by injecting the modified RABV into V1 of nontransplanted WT mice and by transplanting cells expressing only the TVA receptor and RFP, but no G protein, followed by RABV injection (fig. S3). We only detected double-labeled donor cells (RFP+ /GFP+ ) but no traced presynaptic partners in the aged or APP/PS1 grafted cortices (fig. S3), indicating that the RABV spread is specific to cells expressing the G protein. We then proceeded with the experimental conditions by transplanting cells expressing TVA and G protein together with an FP. Grafted cells that were infected by the RABV are referred to as starter cells and were present within the graft in all conditions [RFP+ /GFP+ ; Fig. 1, B to D′; for the exact number of starter cells per mouse and condition, see fig. S2 (F and G)]. Transplants were surrounded by GFP+ traced neurons, i.e., input neurons (or RFP+ input neurons, depending on the donor cell type and corresponding RABV), in all three conditions. However, these were substantially higher in number in the cortices of transgenic APP/PS1 and WT aged mice (Fig. 1, B to D). Thus, transplanted neurons survive and integrate into these brain environments in the absence of any prior neuronal loss. Excessive local connectivity of neuronal grafts in an amyloid plaque–loaded cortex Next, we quantified the distribution of the traced neurons across the brain and their average distance to the graft core in the visual cortex (Fig. 1, E and F) to control for any changes in long-distance tracing or any unexpected effects in these host environments. Notably, the brain-wide distribution and distance of local traced neurons were indistinguishable between experimental groups (Fig.  1,  E  and  F). We then calculated the brain-wide input connectivity expressed as connectivity ratio (CR) for each and all innervating regions, as described before (18). The number of input neurons in a given brain region was divided by the total number of starter neurons (RFP+ /GFP+ ) within the graft to calculate the CR and to allow comparison between mice and conditions. The number of starter neurons varied between transplants but did not differ systematically between the experimental conditions (fig. S2, F and G). A total of 26 innervating brain regions were mapped (Fig. 2A), all known to innervate V1 (51, 52), indicating the absence of aberrant connectivity in all these conditions. We consider this an important finding for cellbased therapy for AD patients and elderly. Most inputs derived from local visual cortex (Vis) neurons, with the highest CR consistently found for this region (Fig. 2A), as is the case for V1 neurons in the naïve mouse brain (18, 51, 52). The CR for this anatomical region (Vis-to-graft, intra-area) in the control group was very similar to that previously measured for endogenous connections within this area using the same tracing technology and analytical pipeline. In contrast, this local innervation was significantly elevated in APP/PS1 transgenic mice (Fig. 2, A and B), as indicated by a threefold increase in CR. This was not the case for other regions and the main afferent of V1, the thalamic dorsal lateral geniculate nucleus (LGN) (51), with no significant difference in the LGN-to-Vis connectivity in APP/PS1 as compared to WT control brains (Fig. 2, A and C). Notably, the CR for other cortical regions with high plaque deposition, such as RS, PtPa, SS, MO, Aud, ECT, ENT, Tea, and Orb (see abbreviations in Fig. 2A), was comparable between APP/PS1 and control WT brains of equal age and from the same colony and background (Fig. 2D). This was also the case for the contralateral hemisphere (Fig. 2E; for the exact numbers of traced neurons per mouse and brain area, see data S2). Thus, in the amyloid plaque–loaded cortex, the local inputs to the graft are specifically enhanced. This is not related to the plaque load in the innervating region, as relatively normal connectivity from other cortical regions with similar plaque load was observed. Notably, however, statistical significance of CR changes in regions with low CR and hence traced neurons present only in some animals may be missed. 

 

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