Biology subjects

Pasternak, S. H.

Publications and source records attributed to Pasternak, S. H..

6 recordsLinked to original sources

bioRxiv2025

Characterizing a hallmark of glymphatic insufficiency: Wasteosomes accumulate in periventricular white matter hyperintensities and exhibit complex relationships with mixed pathology, sclerotic index and perivascular space

The glymphatic system is a recently elucidated waste clearance system in the brain, thought to be critical for the maintenance of homeostasis. Corpora amylacea or "wasteosomes", are discontinuous lipid labyrinth structures that are polyglucosan rich, retain cellular waste and are thought to be of astrocytic origin. Wasteosomes have been proposed as a hallmark of glymphatic insufficiency predominantly due to: 1) their spatial localization around glymphatic drainage points including periventricular (PV) regions, perivascular spaces (PVS), and sub-pial regions; and, 2) their correlation with aging, vascular disorders, neurodegenerative diseases, and conditions that impair sleep. White matter hyperintensities (WMHs) are diffuse hyperintense areas seen on T2-weighted or fluid-attenuated inversion recovery (FLAIR) magnetic resonance imaging (MRI) scans that represent damage to white matter. PV WMHs and are known predictors of mild cognitive impairment, stroke, dementia and death. The relationship between wasteosome accumulation, PV WMHs, vascular pathology and PVS is currently unknown. For the first time, in a mixed diagnostic cohort of pathologically diagnosed: Alzheimers disease (AD), cerebrovascular disease (CVD), mixed AD/CVD, and control tissue with no pathological diagnosis, we connected the histopathological wasteosome profile in periventricular brain sections in relation to 7T FLAIR-MRI confirmed PV WMHs, vascular stenosis and PVS. Our results reveal wasteosomes accumulate in PV WMHs, are increased in proximity to large PV venules, and exhibit complex relationships with WMH severity, mixed pathology, sclerotic index and PVS. These findings suggest wasteosomes may serve as histological markers of impaired glymphatic drainage and provide new insights into the pathophysiology underlying white matter injury.

neuroscience↗

bioRxiv2025

Increased circulating TREM2+ microglia extracellular vesicles in aged APP/PS1 Alzheimer's disease rats

INTRODUCTIONTREM2 is a microglial marker important in Alzheimers disease (AD) pathogenesis, but current methods to detect microglial TREM2 expression in vivo are limited. Circulating microglia-derived extracellular vesicles (EVs) show promise as potential biomarkers for AD and may offer insight into TREM2 activity. METHODSTMEM119+/TREM2+ EVs were assessed using nanoscale flow cytometry in plasma from wildtype and APP/PS1 rats aged to 3-, 9-, and 15-months-old. Molecular and histological assays were used to assess microglia markers in rat brain tissue and a radial arm water maze task was employed to evaluate spatial working and reference memory. RESULTSCirculating TMEM119+/TREM2+ EVs were increased in 15-month APP/PS1 rats and associated with severity of cognitive impairment. TREM2 brain expression varied by anatomical region, age, transgene, and assay. DISCUSSIONCollectively, this study provides the first assessment of TMEM119+/TREM2+ EVs as a biomarker of brain microglial expression and cognition in an AD rat model.

neuroscience↗

bioRxiv2025

Macropinocytosis of amyloid precursor protein requires the adaptor protein Fe65 and the recruitment and activity of Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA.

Alzheimers disease (AD) is a progressive neurodegenerative disorder characterized by the buildup of the highly toxic peptide amyloid-beta (A{beta}). Previously, we demonstrated that A{beta} is generated from the cleavage of amyloid precursor protein (APP) after internalization to lysosomes via macropinocytosis. However, the regulation of APP micropinocytosis has remained uncharacterized. Evidence suggests that APP may function as a cell surface receptor which could contribute to this regulation. Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA are known to regulate macropinocytosis in response to signaling of other receptors. An adaptor protein called Fe65, which can associate with both amyloid precursor protein and Arf6, could function as the link between APP and these known regulatory elements. Thus, we hypothesized that the binding and/or crosslinking of APP recruits Fe65, which then recruits and activates Arf6, which in turn activates Rac1, Cdc42 and RhoA, resulting in APP macropinocytosis. Rapid and transient recruitment of Fe65 and Arft6 was observed to APP 30 seconds following binding/crosslinking. Rac1, Cdc42 and RhoA all examined demonstrated more sustained recruitment to crosslinked APP. Prevention of Fe65 binding by APP mutation and Arf6 inhibition by NAV-2729 prevented the recruitment of all proteins. Together, these observations are the first to demonstrate that a network of regulatory proteins is recruited to bound/crosslinked APP which regulates its macropinocytosis. Targeting these regulatory proteins could be explored to modulate the membrane to lysosomal trafficking of APP and reducing the production of A{beta} in AD.

neuroscience↗

bioRxiv2025

Macropinocytosis of aggregated amyloid-beta and tau requires Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA.

The neuron-to-neuron transfer of amyloid-beta (A{beta}) and tau aggregates have been proposed to underlie the propagation of protein aggregation in Alzheimers disease (AD) and contributing to progressive neurodegeneration. Several studies have provided evidence that aggregates of A{beta} and tau are taken up into neuronal cells and neurons from the extracellular environment, where they contribute to the propagation of A{beta} and tau aggregation in AD through seeding their aggregation. As a result, attention has been placed on determining the cellular mechanisms that contribute to this uptake, with the hopes that targeting these mechanisms could halt the progression of AD by preventing aggregate transfer. Previous studies have demonstrated the uptake of A{beta} and tau aggregates through the endocytic process called macropinocytosis. The activity of several GTPases has been demonstrated to regulate macropinocytosis, including Arf6 and the RhoGTPases Rac1, Cdc42 and RhoA. Here, we examined the uptake of A{beta}42 oligomers and tau fibrils by macropinocytosis in neurons and the role of Arf6, Rac1, Cdc42 and RhoA activity in the macropinocytosis of these aggregates. In this study, we demonstrated that extracellular A{beta}42 oligomers and tau fibrils are taken up by iPSC-derived neurons and delivered directly to LAMP1-labeled lysosomes through macropinocytosis. This was demonstrated by reduced uptake in response to treatment with the macropinocytosis inhibitor EIPA, but not in response to the clathrin inhibitor Pitstop2. The uptake of these aggregates by macropinocytosis was significantly reduced by the inhibition of the GTPases Arf6, Rac1, Cdc42 and RhoA. Further, we also demonstrated that accumulation of extracellular A{beta}42 oligomers and tau fibrils results in an accumulation of cytoplasmic tau within aggregate-containing lysosomes. Together these results provide evidence that the GTPases Arf6, Rac1, Cdc42 and RhoA play a role in the neuronal uptake of A{beta} and tau aggregates by macropinocytosis and identifies new molecular targets to explore preventing the neuron-to-neuron transfer of these aggregates in AD.

neuroscience↗

bioRxiv2025

Amyloid beta is released by lysosomal exocytosis from hiPSC-derived neurons

The endosomal-lysosomal system has long been linked to the production of amyloid beta (A{beta}), but the specific intracellular compartments involved in A{beta} secretion remain contentious. While lysosomes are typically associated with A{beta} degradation, studies have also shown that lysosomes also serve as site of A{beta} accumulation in cells, mouse models, and human tissues. Lysosomal exocytosis is a major secretory pathway in non-neuronal cells, but few studies have investigated this pathway in neurons. Here, we examined the potential role and mechanism of lysosomal exocytosis in human induced pluripotent stem cell (hiPSC)-derived neurons, and we hypothesized that lysosomal exocytosis is a pathway for A{beta} secretion from these neurons. Using total internal reflection fluorescence (TIRF) microscopy, lysosomes filled with fluorescently labelled amyloid were seen approaching and fusing with the plasma membrane in real-time. The number and composition of the released particles were characterized using nanoscale flow cytometry. Silencing two proteins, Rab27b and munc13-4, significantly reduced these events and blocked the release of amyloid into the extracellular space. Our results provide direct evidence for the involvement of lysosomal exocytosis in the release of A{beta} from neurons and highlight its potential as a target for therapeutic intervention in Alzheimers disease.

neuroscience↗

bioRxiv2020

Ultrafiltration Segregates Tissue Regenerative Stimuli Harboured Within and Independent of Extracellular Vesicles

The release of extracellular vesicles (EVs) from human multipotent stromal cells (MSC) has been proposed as a mechanism by which MSC mediate regenerative functions in vivo. Our recent work has characterized MSC derived from human pancreatic tissues (Panc-MSC) that generated a tissue regenerative secretome. Despite these advancements, it remains unknown whether regenerative stimuli are released independent or within extracellular vesicles. Herein, this study demonstrates ultrafiltration is a simple method to enrich for EVs which can be injected in murine models of tissue regeneration. The enrichment of EVs from Panc-MSC conditioned media (CM) was validated using nanoscale flow cytometry and atomic force microscopy; in addition to the exclusive detection of classical EV-markers CD9, CD81, CD63 using label-free mass spectrometry. Additionally, we identified several pro-regenerative stimuli, such as WNT5A or ANGPT1, exclusive to EV-enriched CM. Endothelial cell tubule formation was enhanced in response to both Panc-MSC CM fractions in vitro yet only intramuscular injection of EV-enriched CM demonstrated vascular regenerative functions in NOD/SCID mice with unilateral hind-limb ischemia (*<p<0.05). Furthermore, both EV-depleted and EV-enriched CM reduced hyperglycemia following intrapancreatic injection in hyperglycemic mice (**p<0.01). Collectively, understanding the functional synergy between compartments of the secretome is required to advance cell-free biotherapeutics into applications of regenerative medicine.

cell biology↗