Poloxamer 188 Attenuatesin vitroTraumatic Brain Injury-Induced Mitochondrial and Lysosomal Membrane Permeabilization Damage in Cultured Primary Neurons

Abstract

Acute membrane damage due to traumatic brain injury (TBI) is a critical precipitating event. However, the subsequent effects of the mechanical trauma, including mitochondrial and lysosomal membrane permeability (MOMP and LMP) remain elusive. The main objective of the current study was to assess the role of a putative membrane-resealing agent poloxamer 188 (P188) in MOMP and LMP in response to a well-defined mechanical insult. Using an in vitro cell shearing device (VCSD), mechanical injury resulted in immediate disruption of membrane integrity in cultured primary neurons, and neurons were treated with P188 or a cathepsin B inhibitor (CBI) after VCSD 10 min. The protective effect of P188 on cultured primary neurons was first detected visually with a light microscope, and measured by MTT assay and LDH assay. The validity of monitoring changes in mitochondrial membrane potential (ΔΨm) was measured by JC-1 staining, and Western blot for cytochrome c and truncated Bid (tBid) in purified mitochondria was also performed. In addition, lysosomal integrity was detected by blotting for cathepsin B and tBid in purified lysosomes. Our results showed post-injury P188 treatment moderated the dissipation of ΔΨm in mitochondria, and inhibited VCSD-induced cytochrome c release from mitochondria as well as cathepsin B from lysosomes. Cathepsin B inhibition (CBI) could also increase cell viability, maintain mitochondrial membrane potential, and repress VCSD-induced release of cytochrome c from mitochondria to cytosol. Both P188 and CBI treatment decreased the cytosolic accumulation of tBid in supernatant of purified lysosomes, and the amount of mitochondrial localized tBid. These data indicate injured neurons have undergone mitochondrial and lysosomal membrane permeability damage, and the mechanism can be exploited with pharmacological interventions. P188's neuroprotection appears to involve a relationship between cathepsin B and tBid-mediated mitochondrial initiation of cell death.

The Cysteine Protease Cathepsin B Is a Key Drug Target and Cysteine Protease Inhibitors Are Potential Therapeutics for Traumatic Brain Injury

“…56 Moreover, cathepsin B leakage from lysosomes to the cytoplasm has been shown to occur in an in vitro neuronal cell system, which models TBI injury. 57 Thus, TBIinduced lysosomal leakage of cathepsin B into the cytoplasm may be a key event in causing neuronal cell death.…”

Neuropeptide Y damages the integrity of mitochondrial structure and disrupts energy metabolism in cultured neonatal rat cardiomyocytes

“…Cultured neonatal rat cardiomyocytes were incubated with different dose of NPY for 36 h. 5 ml of Cardiomyocytes (10 6 /ml) were collected and fixed with 2.5% glutaraldehyde, in 0.1 M cacodylate buffer at pH 7.2 for 12 h and postfixed with 1% osmic acid in the cacodylate buffer for 1 h as previously described [22]. Samples were routinely dehydrated in grade ethyl alcohol and propylene oxide, and then embeded in a mixture of epoxy resin and cured overnight at 60 • C. Sections (70 nm thick) were cut with glass knife on an ultracut microtome, stained with uranyl acetate and lead citrate, and examined with Philips CM-120 electron microscope at an accelerating voltage of 120 kV after staining with uranyl acetate and lead citrate.…”

“…Samples were routinely dehydrated in grade ethyl alcohol and propylene oxide, and then embeded in a mixture of epoxy resin and cured overnight at 60 • C. Sections (70 nm thick) were cut with glass knife on an ultracut microtome, stained with uranyl acetate and lead citrate, and examined with Philips CM-120 electron microscope at an accelerating voltage of 120 kV after staining with uranyl acetate and lead citrate. Quantitative morphometric analysis of mitochondrion was conducted according to the method as previously described [22].…”

“…The loss of cell mitochondrial integrity has recently been found to be a major contributor to the development of cellular damage, and consequently leading to ionic imbalances and activation of several other cellular pathways [19,22]. The fluorescent probe 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethyl benzimidazolocarbocyanine iodide (JC-1) was used to investigate mitochondrial membrane potential ( m) in primary cells [22].…”

“…The loss of cell mitochondrial integrity has recently been found to be a major contributor to the development of cellular damage, and consequently leading to ionic imbalances and activation of several other cellular pathways [19,22]. The fluorescent probe 5,5 ,6,6 -tetrachloro-1,1 ,3,3 -tetraethyl benzimidazolocarbocyanine iodide (JC-1) was used to investigate mitochondrial membrane potential ( m) in primary cells [22]. JC-1 enters the mitochondria as a monomer, forms aggregates that have a fluorescence emission spectra peak at 590 nm at high m (red), and becomes a monomer again with a fluorescence emission peak at 529 nm (green) at low m. Peroxisome proliferator-activated receptor ␥ coactivator1alpha (PGC-1␣) forms part of the molecular regulatory circuitry involved in the transcriptional control of cellular energy metabolism, especially in mitochondrial function and biogenesis [37].…”