Real-time heterogeneity of supramolecular assembly of amyloid precursor protein is modulated by an endocytic risk factor PICALM.

Recently, the localization of amyloid precursor protein (APP) into reversible nanoscale supramolecular assembly or "nanodomains" has been highlighted as crucial towards understanding the onset of the molecular pathology of Alzheimer's disease (AD). Surface expression of APP is regulated by proteins interacting with it, controlling its retention and lateral trafficking on the synaptic membrane. Here, we evaluated the involvement of a key risk factor for AD, PICALM, as a critical regulator of nanoscale dynamics of APP. Although it was enriched in the postsynaptic density, PICALM was also localized to the presynaptic active zone and the endocytic zone. PICALM colocalized with APP and formed nanodomains with distinct morphological properties in different subsynaptic regions. Next, we evaluated if this localization to subsynaptic compartments was regulated by the C-terminal sequences of APP, namely, the "Y682ENPTY687" domain. Towards this, we found that deletion of C-terminal regions of APP with partial or complete deletion of Y682ENPTY687, namely, APP-Δ9 and APP-Δ14, affected the lateral diffusion and nanoscale segregation of APP. Lateral diffusion of APP mutant APP-Δ14 sequence mimicked that of a detrimental Swedish mutant of APP, namely, APP-SWE, while APP-Δ9 diffused similar to wild-type APP. Interestingly, elevated expression of PICALM differentially altered the lateral diffusion of the APP C-terminal deletion mutants. These observations confirm that the C-terminal sequence of APP regulates its lateral diffusion and the formation of reversible nanoscale domains. Thus, when combined with autosomal dominant mutations, it generates distinct molecular patterns leading to onset of Alzheimer's disease (AD).

The delayed kinetics of Myddosome formation explains why amyloid-beta aggregates trigger Toll-like receptor 4 less efficiently than lipopolysaccharide.

The Myddosome is a key innate immune signalling platform. It forms at the cell surface and contains MyD88 and IRAK proteins which ultimately coordinate the production of pro-inflammatory cytokines. Toll-like receptor 4 (TLR4) signals via the Myddosome when triggered by lipopolysaccharide (LPS) or amyloid-beta (Aß) aggregates but the magnitude and time duration of the response are very different for reasons that are unclear. Here, we followed the formation of Myddosomes in live macrophages using local delivery of TLR4 agonist to the cell surface and visualisation with 3D rapid light sheet imaging. This was complemented by super-resolution imaging of Myddosomes in fixed macrophages to determine the size of the signalling complex at different times after triggering. Myddosomes formed more rapidly after LPS than in response to sonicated Aß 1-42 fibrils (80 vs 372 s). The mean lifetimes of the Myddosomes were also shorter when triggered by LPS compared to sonicated Aß fibrils (170 and 220 s), respectively. In both cases, a range of Myddosome of different sizes (50-500 nm) were formed. In particular, small round Myddosomes around 100 nm in size formed at early time points, then reduced in proportion over time. Collectively, our data suggest that compared to LPS the multivalency of Aß fibrils leads to the formation of larger Myddosomes which form more slowly and, due to their size, take longer to disassemble. This explains why sonicated Aß fibrils results in less efficient triggering of TLR4 signalling and may be a general property of protein aggregates.

Golgi fragmentation - One of the earliest organelle phenotypes in Alzheimer's disease neurons.

Alzheimer's disease (AD) is the most common cause of dementia, with no current cure. Consequently, alternative approaches focusing on early pathological events in specific neuronal populations, besides targeting the well-studied amyloid beta (Aß) accumulations and Tau tangles, are needed. In this study, we have investigated disease phenotypes specific to glutamatergic forebrain neurons and mapped the timeline of their occurrence, by implementing familial and sporadic human induced pluripotent stem cell models as well as the 5xFAD mouse model. We recapitulated characteristic late AD phenotypes, such as increased Aß secretion and Tau hyperphosphorylation, as well as previously well documented mitochondrial and synaptic deficits. Intriguingly, we identified Golgi fragmentation as one of the earliest AD phenotypes, indicating potential impairments in protein processing and post-translational modifications. Computational analysis of RNA sequencing data revealed differentially expressed genes involved in glycosylation and glycan patterns, whilst total glycan profiling revealed minor glycosylation differences. This indicates general robustness of glycosylation besides the observed fragmented morphology. Importantly, we identified that genetic variants in Sortilin-related receptor 1 (SORL1) associated with AD could aggravate the Golgi fragmentation and subsequent glycosylation changes. In summary, we identified Golgi fragmentation as one of the earliest disease phenotypes in AD neurons in various in vivo and in vitro complementary disease models, which can be exacerbated via additional risk variants in SORL1.

Quantifying molecular aggregation by super resolution microscopy within an excitatory synapse from mouse hippocampal neurons.

Super-resolution microscopy (SRM) has been widely adopted to probe molecular distribution at excitatory synapses. We present an SRM paradigm to evaluate the nanoscale organization heterogeneity between neuronal subcompartments. Using mouse hippocampal neurons, we describe the identification of the morphological characteristics of nanodomains within functional zones of a single excitatory synapse. This information can be used to correlate structure and function at molecular resolution in single synapses. The protocol can be applied to immunocytochemical/histochemical samples across different imaging paradigms. For complete details on the use and execution of this protocol, please refer to Kedia et al. (2021).

Nanoscale organization of Nicastrin, the substrate receptor of the γ-secretase complex, as independent molecular domains.

Alterations in the canonical processing of Amyloid Precursor Protein generate proteoforms that contribute to the onset of Alzheimer's Disease. Modified composition of γ-secretase or mutations in its subunits has been directly linked to altered generation of Amyloid beta. Despite biochemical evidence about the role of γ-secretase in the generation of APP, the molecular origin of how spatial heterogeneity in the generation of proteoforms arises is not well understood. Here, we evaluated the localization of Nicastrin, a γ-secretase subunit, at nanometer sized functional zones of the synapse. With the help of super resolution microscopy, we confirm that Nicastrin is organized into nanodomains of high molecular density within an excitatory synapse. A similar nanoorganization was also observed for APP and the catalytic subunit of γ-secretase, Presenilin 1, that were discretely associated with Nicastrin nanodomains. Though Nicastrin is a functional subunit of γ-secretase, the Nicastrin and Presenilin1 nanodomains were either colocalized or localized independent of each other. The Nicastrin and Presenilin domains highlight a potential independent regulation of these molecules different from their canonical secretase function. The collisions between secretases and substrate molecules decide the probability and rate of product formation for transmembrane proteolysis. Our observations of secretase nanodomains indicate a spatial difference in the confinement of substrate and secretases, affecting the local probability of product formation by increasing their molecular availability, resulting in differential generation of proteoforms even within single synapses.

Alteration in synaptic nanoscale organization dictates amyloidogenic processing in Alzheimer's disease.

Despite intuitive insights into differential proteolysis of amyloid precursor protein (APP), the stochasticity behind local product formation through amyloidogenic pathway at individual synapses remain unclear. Here, we show that the major components of amyloidogenic machinery namely, APP and secretases are discretely organized into nanodomains of high local concentration compared to their immediate environment in functional zones of the synapse. Additionally, with the aid of multiple models of Alzheimer's disease (AD), we confirm that this discrete nanoscale chemical map of amyloidogenic machinery is altered at excitatory synapses. Furthermore, we provide realistic models of amyloidogenic processing in unitary vesicles originating from the endocytic zone of excitatory synapses. Thus, we show how an alteration in the stochasticity of synaptic nanoscale organization contributes to the dynamic range of C-terminal fragments ß (CTFß) production, defining the heterogeneity of amyloidogenic processing at individual synapses, leading to long-term synaptic deficits as seen in AD.

Nanoscale rearrangement of APP organization as a therapeutic target for Alzheimer's disease.

Despite the importance of canonical processing of Amyloid Precursor Protein at synapses as a major risk factor for the development of Alzheimer's Disease, there have been very little progress on designing effective therapeutic paradigms targeting it. Majority of the drugs developed or under clinical evaluation focus on the clearance of the detrimental proteoforms or secretases involved in the proteolysis of APP. The lack of interventions targeting APP is in part due to the lack of information in understanding the fine organization of APP and the chemical map of its association with subsynaptic functional zones of the synapse. The recent advances to evaluate the molecular organization of synapses allows us to readdress the need for designing tools to target the full-length APP. Here, we describe the potential role of nanoscale segregation of synaptic APP and how this organization influences the local processing of APP in different subsynaptic compartments opening avenues for early intervention strategies. We envision the need to design smart molecules which would interfere with the real-time chemical composition and physical properties of APP at nanoscale. These tools could alter the balance of proteoforms generated and/or enhance the proteolysis by selective secretases to reduce the toxic products formed through amyloidogenic pathway. We believe that such an approach would be rational to treat or delay the onset of neurodegenerative diseases like AD.

Real-time nanoscale organization of amyloid precursor protein.

Despite an intuitive understanding of the role of APP in health and disease, there exist few attempts to dissect its molecular localization at excitatory synapses. Though the biochemistry involved in the enzymatic processing of APP is well understood, there is a void in understanding the nonuniformity of the product formation in vivo. Here, we employed multiple paradigms of single molecules and ensemble based nanoscopic imaging to reveal that APP molecules are organized into regulatory nanodomains that are differentially compartmentalized in the functional zones of an excitatory synapse. Furthermore, with the aid of high density single particle tracking, we show that the lateral diffusion of APP in live cells dictates an equilibrium between these nanodomains and their nano-environment, which is affected in a detrimental variant of APP. Additionally, we incorporate this spatio-temporal detail 'in silico' to generate a realistic nanoscale topography of APP in dendrites and synapses. This approach uncovers a nanoscale heterogeneity in the molecular organization of APP, depicting a locus for differential APP processing. This holistic paradigm, to decipher the real-time heterogeneity of the substrate molecules on the nanoscale, could enable us to better evaluate the molecular constraints overcoming the ensemble approaches used traditionally to understand the kinetics of product formation.