Augello, C. | Search bioRxiv

bioRxiv2021

Spatial Analysis of Neural Cell Proteomic Profiles following Ischemic Stroke in Mice using High-Plex Digital Spatial Profiling

Stroke is ranked as the fifth leading cause of death and the leading cause of adult disability. The progression of neuronal damage after stroke is recognized to be a complex integration of glia, neurons, and the surrounding extracellular matrix, therefore potential treatments must target the detrimental effects created by these interactions. In this study, we examined the spatial cellular and neuroinflammatory mechanisms occurring early after ischemic stroke utilizing Nanostring Digital Spatial Profiling (DSP) technology. Male C57bl/6 mice were subjected to photothrombotic middle cerebral artery occlusion (MCAO) and sacrificed at three-days post-ischemia. Spatial distinction of the ipsilateral hemisphere was studied according to the regions of interest: the ischemic core, peri-infarct tissues, and peri-infarct normal tissue (PiNT) in comparison to the contralateral hemisphere. We demonstrated that the ipsilateral hemisphere initiates distinct spatial regulatory proteomic profiles with DSP technology that can be identified consistently with the immunohistochemical markers, FJB, GFAP, and Iba-1. The core border profile demonstrated an induction of neuronal death, apoptosis, autophagy, immunoreactivity, and early degenerative proteins. Most notably, the core border resulted in a decrease of the neuronal proteins Map2 and NeuN, an increase in the autophagy proteins BAG3 and CTSD, an increase in the microglial and peripheral immune invasion proteins Iba1, CD45, CD11b, and CD39, and an increase in the neurodegenerative proteins BACE1, APP, {beta} 1-42, ApoE, and hyperphosphorylated tau protein S-199. The peri-infarct region demonstrated increased astrocytic immunoreactivity, apoptotic, and neurodegenerative proteomic profile, with an increase in BAG3, GFAP, and hyperphosphorylated tau protein S-199. The PiNT region displayed minimal changes compared to the contralateral cortex with only an increase in GFAP. Overall, our data highlight the importance of identifying ischemic mechanisms in a spatial manner to understand the complex, dynamic interactions throughout ischemic progression and repair as well to introduce potential targets for successful therapeutic interventions.

neuroscience↗

bioRxiv2019

Spontaneous isomerization of long-lived proteins provides a molecular mechanism for the lysosomal failure observed in Alzheimer’s disease

Proteinaceous aggregation is a well-known observable in Alzheimers disease (AD), but failure and storage of lysosomal bodies within neurons is equally ubiquitous and actually precedes bulk accumulation of extracellular amyloid plaque. In fact, AD shares many similarities with certain lysosomal storage disorders though establishing a biochemical connection has proven difficult. Herein, we demonstrate that isomerization and epimerization, which are spontaneous chemical modifications that occur in long-lived proteins, prevent digestion by the proteases in the lysosome (namely the cathepsins). For example, isomerization of aspartic acid into L-isoAsp prevents digestion of the N-terminal portion of A{beta} by cathepsin L, one of the most aggressive lysosomal proteases. Similar results were obtained after examination of various target peptides with a full series of cathepsins, including endo-, amino-, and carboxy-peptidases. In all cases peptide fragments too long for transporter recognition or release from the lysosome persisted after treatment, providing a mechanism for eventual lysosomal storage and bridging the gap between AD and lysosomal storage disorders. Additional experiments with microglial cells confirmed that isomerization disrupts proteolysis in active lysosomes. These results are easily rationalized in terms of protease active sites, which are engineered to precisely orient the peptide backbone and cannot accommodate the backbone shift caused by isoaspartic acid or side chain dislocation resulting from epimerization. Although A{beta} is known to be isomerized and epimerized in plaques present in AD brains, we further establish that the rates of modification for aspartic acid in positions 1 and 7 are fast and could accrue prior to plaque formation. Spontaneous chemistry can therefore provide modified substrates capable of inducing gradual lysosomal failure, which may play an important role in the cascade of events leading to the disrupted proteostasis, amyloid formation, and tauopathies associated with AD.

biochemistry↗