Biology subjects

Sharif, A.

Publications and source records attributed to Sharif, A..

6 recordsLinked to original sources

bioRxiv2026

In differentiated HL-60 neutrophil-like cells, MRP1- (ABCC1-) mediated glutathione efflux stimulated by BzATP and P2X7 receptor signalling regulates exosome release through nSMase activity

Exosomes are endosome-derived extracellular vesicles (EVs) playing key roles in immune regulation and inflammatory signalling, yet with poorly defined mechanisms of biogenesis. We investigated how purinergic signalling and redox regulation intersect, controlling exosome formation in neutrophil-like cells (NLCs) differentiated from the HL-60 cell line. Differentiated NLCs displayed hallmark neutrophil features, including increased CD11b expression, reduced CD71, enhanced phagocytosis, robust reactive oxygen species generation, and cell-cycle arrest. CD63-positive exosomes released from NLCs fulfilled MISEV 2023 criteria, exhibiting typical morphology, density, and size distribution. Stimulation with the P2X7R agonist BzATP induced a rapid, dose-dependent increase in exosome release correlating strongly with depletion of intracellular glutathione (iGSH) and activation of neutral sphingomyelinase (nSMase). Pharmacological inhibition of nSMase abrogated BzATP-induced exosome release, implicating ceramide-dependent intraluminal vesicle formation. Mechanistically, BzATP activated PI3K/AKT signalling, leading to stimulation of the ABC transporter MRP1/ABCC1, enhanced efflux of GSH, and a concomitant rise in extracellular GSH. Inhibition of PI3K/AKT or MRP1 prevented iGSH efflux, reduced nSMase activity, and significantly attenuated exosome secretion. These findings define a previously uncharacterized pathway in NLCs whereby extracellular ATP acts as a danger signal triggering PI3K/AKT-MRP1-dependent redox gating of nSMase activity, thereby driving rapid exosome biogenesis. This mechanism provides insight into how neutrophils translate inflammatory cues into vesicle-mediated communication and highlights potential therapeutic targets to modulate pathological neutrophil-driven inflammation.

cell biology↗

bioRxiv2026

Ultrastructural and Histological Cryopreservation of Mammalian Brains by Vitrification

Studies of whole brain cryopreservation are rare but are potentially important for a variety of applications. It has been demonstrated that ultrastructure in whole rabbit and pig brains can be cryopreserved by vitrification (ice-free cryopreservation) after prior aldehyde fixation, but fixation limits the range of studies that can be done by neurobiologists, including studies that depend upon general molecular integrity, signal transduction, macromolecular synthesis, and other physiological processes. We now show that whole brain ultrastructure can be preserved by vitrification without prior aldehyde fixation. Rabbit brain perfusion with the M22 vitrification solution followed by vitrification, warming, and fixation showed an absence of visible ice damage and overall structural preservation, but osmotic brain shrinkage sufficient to distort and obscure neuroanatomical detail. Neuroanatomical preservation in the presence of M22 was also investigated in human cerebral cortical biopsies taken after whole brain perfusion with M22. These biopsies did not form ice upon cooling or warming, and high power electron microscopy showed dehydrated and electron-dense but predominantly intact cells, neuropil, and synapses with no signs of ice crystal damage, and partial dilution of these samples restored normal cortical pyramidal cell shapes. To further evaluate ultrastructural preservation within the severely dehydrated brain, rabbit brains were perfused with M22 and then partially washed free of M22 before fixation. Perfusion dilution of the brain to 3-5M M22 resulted in brain re-expansion and the re-appearance of well-defined neuroanatomical features, but rehydration of the brain to 1M M22 resulted in ultrastructural damage suggestive of preventable osmotic injury caused by incomplete removal of M22. We conclude that both animal and human brains can be cryopreserved by vitrification with predominant retention of ultrastructural integrity without the need for prior aldehyde fixation. This observation has direct relevance to the feasibility of human cryopreservation, for which direct evidence has been lacking until this report. It also provides a starting point for perfecting brain cryopreservation, which may be necessary for lengthy space travel and could allow future medical time travel.

neuroscience↗

bioRxiv2024

Effects of HA1 (a Probucol Analogue) and ApoC3 siRNA on Lipoprotein-Amyloid Metabolism, Neurovascular Integrity and Cognitive Function in db/db Mice

Background and aimsChronically elevated levels of circulating lipoprotein-amyloid-{beta} (A{beta}) are implicated in the disruption of the blood-brain barrier and the initiation of a neurodegenerative cascade leading to Alzheimers disease (AD). Type 2 diabetes is associated with BBB dysfunction, dyslipidaemia, and an increased risk of AD. However, alterations in triglyceride-rich lipoproteins (TRL)-A{beta} homeostasis and its downstream effects on the BBB in a diabetes context remain explored. This study aimed to 1) investigate, in a preclinical model of diabetes, the hypothesis that diabetes-induced impairments in TRL-A{beta} metabolism might compromise BBB integrity and exacerbate cognitive function and behavioural changes, and 2) assess the efficacy of interventions that improve TRL catabolism, including probucol, HA1, and ApoC3 siRNA, to prevent disease progression by lowering circulating TRL-A{beta} levels. MethodsFive-week-old db/db mice underwent a 9- or 23-weeks dietary interventions with probucol, a probucol prodrug HA1, or a standard diet with four-weekly injections of ApoC3 siRNA. Db/+ mice served as negative controls for each treatment duration. Blood levels of A{beta} and ApoB were measured using ELISA. Immunofluorescence imaging was used to quantify enterocytic levels of A{beta} and ApoB, and assess changes in neurovascular integrity (IgG, PDGFR{beta}, ZO1, occludin), neuroinflammation (GFAP, Iba1), and cerebral oxidative stress (8OHdG). ResultsOur results indicate that diabetes increased the abundance of plasma amyloid, specifically A{beta}42, which correlated with enterocytic abundance, suggesting exaggerated postprandial excretion. Disrupted plasma amyloid homeostasis was associated with BBB breakdown, including diminished barrier function, and the loss of pericytes and astrocytes. Provision of the probucol analogue, HA1, normalised plasma and enterocytic amyloidemia concomitant with the preservation of the neurovascular junction. Treatment with ApoC3 siRNA attenuated plasma A{beta}42 and modestly reduced neurovascular inflammation. ConclusionThe findings further support the hypothesis that aberrant peripheral metabolism of lipoprotein-A{beta} is associated with microvascular corruption and the development of anxiety-like behaviour. HA1 is more effective than probucol or ApoC3 siRNA in positively modulating lipoprotein-amyloid homeostasis in db/db mice and maintaining central capillary integrity. HighlightsO_LIEnterocytic and plasma lipoprotein-A{beta} levels are increased in diabetic db/db mice. C_LIO_LIElevated plasma lipoprotein-A{beta} levels correlate with BBB breakdown and anxiety-like behavior. C_LIO_LIHA1 lowers enterocytic and plasma A{beta} levels, protects BBB, alleviates oxidative stress and anxiety. C_LIO_LIApoC3 siRNA lowers plasma lipoprotein-A{beta}, mitigates neuroinflammation and anxiety. C_LI

neuroscience↗

bioRxiv2024

CCR5 antagonists as neuroprotective and stroke recovery enhancing agents: a preclinical systematic review and meta-analysis

C-C chemokine receptor type 5 (CCR5) antagonists may improve both acute stroke outcome and long-term recovery. Despite their evaluation in ongoing clinical trials, gaps remain in the evidence supporting their use. With a panel of patients with lived experiences of stroke, we performed a systematic review of animal models of stroke that administered a CCR5 antagonist and assessed infarct size or behavioural outcomes. MEDLINE, Web of Science, and Embase were searched. Article screening and data extraction were completed in duplicate. We pooled outcomes using random effects meta-analyses. We assessed risk of bias using the Systematic Review Centre for Laboratory Animal Experimentation (SYRCLE) tool and alignment with the Stroke Treatment Academic Industry Roundtable (STAIR) and Stroke Recovery and Rehabilitation Roundtable (SRRR) recommendations. Five studies representing 10 experiments were included. CCR5 antagonists reduced infarct volume (standard mean difference -1.02; 95% confidence interval -1.58 to -0.46) when compared to stroke-only controls. Varied timing of CCR5 administration (pre- or post-stroke induction) produced similar benefit. CCR5 antagonists significantly improved 11 of 16 behavioural outcomes reported. High risk of bias was present in all studies and critical knowledge gaps in the preclinical evidence were identified using STAIR/SRRR. CCR5 antagonists demonstrate promise; however, rigorously designed preclinical studies that better align with STAIR/SRRR recommendations and downstream clinical trials are warranted. Prospective Register of Systematic Reviews (PROSPERO CRD42023393438).

neuroscience↗

bioRxiv2020

Deletion of INPP5E in the murine retina impairs axoneme formation and prevents photoreceptor disc morphogenesis

INPP5E (pharbin) is a ubiquitously-expressed, farnesylated phosphatidylinositol polyphosphate 5-phosphatase which modulates the phosphoinositide composition of membranes. INPP5E resides in primary cilia, and mutations or loss of INPP5E are associated with ciliary dysfunction. INPP5E missense mutations of the phosphatase catalytic domain cause Joubert syndrome in humans, a syndromic ciliopathy affecting multiple tissues including brain, liver, kidney and retina. We show that, differing from other primary cilia, INPP5E is present in the wildtype photoreceptor inner segment and absent in the outer segment--a modified primary cilium dedicated to phototransduction. We generated Inpp5eF/F;Six3Cre (in short, retInpp5e-/-) mice which exhibit a rapidly progressing rod-cone degeneration nearly completed by postnatal day 21 (P21) in the central retina. Mutant cone outer segments contain vesicles instead of discs as early as P8. While P10 mutant outer segments contain phototransduction and structural proteins, they do not form axonemes and fail to elaborate disc membranes. Connecting cilia of retInpp5e-/- rods appear normal, although IFT-B/A particles accumulate at their distal ends suggesting disrupted intraflagellar transport. These results show that ablation of INPP5E does not impair the secretory pathway responsible for delivery of outer segment-specific proteins, but blocks axonemal extension and prevents disc morphogenesis.

neuroscience↗

bioRxiv2019

Chronic corticosterone administration induces negative valence and impairs positive valence behaviors in mice

Behavioral approaches utilizing rodents to study mood disorders have focused primarily on negative valence behaviors associated with potential threat (anxiety). However, for disorders such as depression, positive valence behaviors that assess reward processing may be more translationally-valid and predictive of antidepressant treatment outcome. Chronic corticosterone (CORT) administration is a well-validated pharmacological stressor that increases negative valence behaviors (David et al., 2009; Gourley et al., 2008a,b; Gourley et al., 2012; Olausson et al., 2013). However, whether chronic stress paradigms such as CORT administration also lead to deficits in positive valence behaviors remains unclear. We treated male C57BL/6J mice with chronic CORT and assessed both negative and positive valence behaviors. We found that CORT induced negative valence behaviors associated with anxiety in the open field and NSF. Interestingly, CORT also impaired instrumental acquisition, reduced sensitivity to a devalued outcome, reduced breakpoint in progressive ratio, and impaired performance in probabilistic reversal learning. Taken together, these results demonstrate that chronic CORT administration at the same dosage both induces negative valence behaviors associated with anxiety and impairs positive valence behaviors associated with reward processing. These data suggest that CORT administration is a useful experimental system for preclinical approaches to studying stress-induced mood disorders.\n\nSignificance StatementChronic exposure to stress can precipitate mood disorders such as anxiety and depression. However, most studies focus on the effects of chronic stress on increasing negative affect behaviors. Elucidating how chronic stress impacts translationally-valid positive valence behaviors is less studied. Here, we show that chronic pharmacological stress induces negative affect behaviors associates with anxiety and impairs reward-related, positive valence behaviors in mice.

neuroscience↗