Caffeic acid phenethyl ester inhibits neuro-inflammation and oxidative stress following spinal cord injury by mitigating mitochondrial dysfunction via the SIRT1/PGC1α/DRP1 signaling pathway.

BACKGROUND: The treatment of spinal cord injury (SCI) has always been a significant research focus of clinical neuroscience, with inhibition of microglia-mediated neuro-inflammation as well as oxidative stress key to successful SCI patient treatment. Caffeic acid phenethyl ester (CAPE), a compound extracted from propolis, has both anti-inflammatory and anti-oxidative effects, but its SCI therapeutic effects have rarely been reported. METHODS: We constructed a mouse spinal cord contusion model and administered CAPE intraperitoneally for 7 consecutive days after injury, and methylprednisolone (MP) was used as a positive control. Hematoxylin-eosin, Nissl, and Luxol Fast Blue staining were used to assess the effect of CAPE on the structures of nervous tissue after SCI. Basso Mouse Scale scores and footprint analysis were used to explore the effect of CAPE on the recovery of motor function by SCI mice. Western blot analysis and immunofluorescence staining assessed levels of inflammatory mediators and oxidative stress-related proteins both in vivo and in vitro after CAPE treatment. Further, reactive oxygen species (ROS) within the cytoplasm were detected using an ROS kit. Changes in mitochondrial membrane potential after CAPE treatment were detected with 5,5',6,6'-tetrachloro-1,1',3,3'-tetraethyl-imidacarbocyanine iodide. Mechanistically, western blot analysis and immunofluorescence staining were used to examine the effect of CAPE on the SIRT1/PGC1α/DRP1 signaling pathway. RESULTS: CAPE-treated SCI mice showed less neuronal tissue loss, more neuronal survival, and reduced demyelination. Interestingly, SCI mice treated with CAPE showed better recovery of motor function. CAPE treatment reduced the expression of inflammatory and oxidative mediators, including iNOS, COX-2, TNF-α, IL-1ß, 1L-6, NOX-2, and NOX-4, as well as the positive control MP both in vitro and in vivo. In addition, molecular docking experiments showed that CAPE had a high affinity for SIRT1, and that CAPE treatment significantly activated SIRT1 and PGC1α, with down-regulation of DRP1. Further, CAPE treatment significantly reduced the level of ROS in cellular cytoplasm and increased the mitochondrial membrane potential, which improved normal mitochondrial function. After administering the SIRT1 inhibitor nicotinamide, the effect of CAPE on neuro-inflammation and oxidative stress was reversed.On the contrary, SIRT1 agonist SRT2183 further enhanced the anti-inflammatory and antioxidant effects of CAPE, indicating that the anti-inflammatory and anti-oxidative stress effects of CAPE after SCI were dependent on SIRT1. CONCLUSION: CAPE inhibits microglia-mediated neuro-inflammation and oxidative stress and supports mitochondrial function by regulating the SIRT1/PGC1α/DRP1 signaling pathway after SCI. These effects demonstrate that CAPE reduces nerve tissue damage. Therefore, CAPE is a potential drug for the treatment of SCI through production of anti-inflammatory and anti-oxidative stress effects.

A Cholecystokinin Analogue Ameliorates Cognitive Deficits and Regulates Mitochondrial Dynamics via the AMPK/Drp1 Pathway in APP/PS1 Mice.

BACKGROUND: There are no drugs on the market that can reverse or slow Alzheimer's disease (AD) progression. A protease-resistant Cholecystokinin (CCK) analogue used in this study is based on the basic structure of CCK, which further increases the stability of the peptide fragment and prolongs its half-life in vivo. We observed a neuroprotective effect of CCK-8L in APPswe/PS1dE9 (APP/PS1) AD mice. However, its corresponding mechanisms still need to be elucidated. OBJECTIVE: This study examined CCK-8L's neuroprotective effects in enhancing cognitive impairment by regulating mitochondrial dynamics through AMPK/Drp1 pathway in the APP/PS1 AD mice. METHODS: Behavioural tests are applied to assess competence in cognitive functions. Transmission electron microscopy (TEM) was performed to observe the ultrastructure of mitochondria of hippocampal neurons, Immunofluorescent staining was employed to assay for Aß1-42, APP, Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and dynamin-related protein1 (Drp1). CRISPR/Cas9 was utilized for targeted knockout of the CCKB receptor (CCKBR) in the mouse APP/PS1 hippocampal CA1 region. A model of lentiviral vector-mediated overexpression of APP in N2a cells was constructed. RESULTS: In vivo, experiments revealed that CCK analogue and liraglutide significantly alleviated cognitive deficits in APP/PS1 mice, reduced Aß1-42 expression, and ameliorated l damage, which is associated with CCKBR activation in the hippocampal CA1 region of mice. In vitro tests showed that CCK inhibited mitochondrial fission and promoted fusion through AMPK/Drp1 pathway. CONCLUSIONS: CCK analogue ameliorates cognitive deficits and regulates mitochondrial dynamics by activating the CCKB receptor and the AMPK/Drp1 pathway in AD mice.

Dexmedetomidine ameliorates high glucose-induced epithelial-mesenchymal transformation in HK-2 cells through the Cdk5/Drp1/ROS pathway.

Epithelial-mesenchymal transformation (EMT) plays an important role in the progression of diabetic nephropathy. Dexmedetomidine (DEX) has shown renoprotective effects against ischemic reperfusion injury; however, whether and how DEX prevents high glucose-induced EMT in renal tubular epithelial cells is incompletely known. Here, we conduct in vitro experiments using HK-2 cells, a human tubular epithelial cell line. Our results demonstrate that high glucose increases the expressions of EMT-related proteins, including Vimentin, Slug, Snail and Twist, while decreasing the expression of E-cadherin and increasing Cdk5 expression in HK-2 cells. Both Cdk5 knockdown and inhibition by roscovitine increase the expressions of E-cadherin while decreasing the expressions of other EMT-related markers. DEX inhibits Cdk5 expression without affecting cell viability and changes the expressions of EMT-related markers, similar to effects of Cdk5 inhibition. Furthermore, Cdk5 is found to interact with Drp1 at the protein level and mediate the phosphorylation of Drp1. In addition, Drp1 inhibition with mdivi-1 could also restrain the high glucose-induced EMT process in HK-2 cells. Immunofluorescence results show that roscovitine, Mdivi-1 and DEX inhibit high glucose-induced intracellular ROS accumulation, while the oxidant H 2O 2 eliminates the protective effect of DEX on the EMT process. These results indicate that DEX mitigates high glucose-induced EMT progression in HK-2 cells via inhibition of the Cdk5/Drp1/ROS pathway.

Bromodomain-containing protein 4 inhibitor JQ1 promotes melanoma cell apoptosis by regulating mitochondrial dynamics.

Although the role of bromodomain-containing protein 4 (BRD4) in ovarian cancer, pancreatic cancer, lymphoma, and many other diseases is well known, its function in cutaneous melanoma is only partially understood. The results of the present study show that the BRD4 inhibitor JQ1 promotes the apoptosis of B16 melanoma cells by altering mitochondrial dynamics, thereby inducing mitochondrial dysfunction and increasing oxidative stress. We found that treatment of B16 cells with different concentrations of JQ1 (125 nmol/L or 250 nmol/L) significantly downregulated the expression of protein subunits involved in mitochondrial respiratory chain complexes I, III, IV, and V, increased reactive oxygen species, induced energy metabolism dysfunction, significantly enhanced apoptosis, and activated the mitochondrial apoptosis pathway. At the same time, JQ1 inhibited the activation of AMP-activated protein kinase, a metabolic energy sensor. In addition, we found that the mRNA and protein levels of mitochondrial dynamin-related protein 1 increased, whereas the levels of mitochondrial fusion protein 1 and optic atrophy protein 1 decreased. Mechanistically, we determined that JQ1 inhibited the expression of c-Myc and altered mitochondrial dynamics, eventually leading to changes in the mitochondrial function, metabolism, and apoptosis of B16 melanoma cells.

Perillyl Alcohol Mitigates Behavioural Changes and Limits Cell Death and Mitochondrial Changes in Unilateral 6-OHDA Lesion Model of Parkinson's Disease Through Alleviation of Oxidative Stress.

In this study, we aim to assess the phytomedicinal potential of perillyl alcohol (PA), a dietary monoterpenoid, in a unilateral 6-hydroxydopamine (6-OHDA) lesion rat model of Parkinson's disease (PD). We observed that PA supplementation alleviated behavioural abnormalities such as loss of coordination, reduced rearing and motor asymmetry in lesioned animals. We also observed that PA-treated animals exhibited reduced oxidative stress, DNA fragmentation and caspase 3 activity indicating alleviation of apoptotic cell death. We found reduced mRNA levels of pro-apoptotic regulator BAX and pro-inflammatory mediators IL18 and TNFα in PA-treated animals. Further, PA treatment successfully increased mRNA and protein levels of Bcl2, mitochondrial biogenesis regulator PGC1α and tyrosine hydroxylase (TH) in lesioned animals. We observed that PA treatment blocked BAX and Drp1 translocation to mitochondria, an event often associated with the inception of apoptosis. Further, 6-OHDA exposure reduced expression of electron transport chain complexes I and IV, thereby disturbing energy metabolism. Conversely, expression levels of both complexes were upregulated with PA treatment in lesioned rats. Finally, we found that protein levels of Nrf2, the transcription factor responsible for antioxidant gene expression, were markedly reduced in cytosolic and nuclear fraction on 6-OHDA exposure, and PA increased expression of Nrf2 in both fractions. We believe that our data hints towards PA having the ability to provide cytoprotection in a hemiparkinsonian rat model through alleviation of motor deficits, oxidative stress, mitochondrial dysfunction and apoptosis.

Muscone suppresses inflammatory responses and neuronal damage in a rat model of cervical spondylotic myelopathy by regulating Drp1-dependent mitochondrial fission.

Cervical spondylotic myelopathy (CSM) is a common cause of disability with few treatments. Aberrant mitochondrial dynamics play a crucial role in the pathogenesis of various neurodegenerative diseases. Thus, regulation of mitochondrial dynamics may offer therapeutic benefit for the treatment of CSM. Muscone, the active ingredient of an odoriferous animal product, exhibits anti-inflammatory and neuroprotective effects for which the underlying mechanisms remain obscure. We hypothesized that muscone might ameliorate inflammatory responses and neuronal damage by regulating mitochondrial dynamics. To this end, the effects of muscone on a rat model of chronic cervical cord compression, as well as activated BV2 cells and injured neurons, were assessed. The results showed that muscone intervention improved motor function compared with vehicle-treated rats. Indeed, muscone attenuated pro-inflammatory cytokine expression, neuronal-apoptosis indicators in the lesion area, and activation of the nod-like receptor family pyrin domain-containing 3 inflammasome, nuclear transcription factor-κB, and dynamin-related protein 1 in Iba1- and ßIII-tubulin-labeled cells. Compared with vehicle-treated rats, compression sites of muscone-treated animals exhibited elongated mitochondrial morphologies in individual cell types and reduced reactive oxygen species. In vitro results indicated that muscone suppressed microglial activation and neuronal damage by regulating related-inflammatory or apoptotic molecules. Moreover, muscone inhibited dynamin-related protein 1 activation in activated BV2 cells and injured neurons, whereby it rescued mitochondrial fragmentation and reactive oxygen species production, which regulate a wide range of inflammatory and apoptotic molecules. Our findings reveal that muscone attenuates neuroinflammation and neuronal damage in rats with chronic cervical cord compression by regulating mitochondrial fission events, suggesting its promise for CSM therapy.

Drp1, a potential therapeutic target for Parkinson's disease, is involved in olfactory bulb pathological alteration in the Rotenone-induced rat model.

Olfaction is often affected in parkinsonian patients and its disturbances precede the classical cognitive and locomotor dysfunction. The olfactory bulb might be the region of onset in Parkinson's disease (PD) pathogenesis, evidenced by the presence of disease-related protein aggregates and disturbed olfactory information processing. However, the underlying molecular mechanism that governs the olfactory bulb impairments remains unclear. This study was designed to investigate the relationship between olfactory bulb and inflammatory pathological alterations and the potential mechanisms. Here we found that rotenone led to typical parkinsonian symptoms and decreased tyrosine hydroxylase (TH)-positive neurons in the olfactory bulb. Additionally, increased NF-κB nuclear translocation and NLRP3 inflammasome components expressions caused by rotenone injection were observed accompanied by the activation of microglia and astrocytes in the olfactory bulb. Rotenone also triggered Drp1-mediated mitochondrial fission and this in turn caused mitochondrial damage. Furthermore, Mdivi-1(a selective Drp1 inhibitor) markedly ameliorated the morphologic disruptions of mitochondria and Drp1 translocation, inhibited the nuclear translocation of NF-κB, eventually blocked the downstream pathway of the NLRP3/caspase-1/IL-1ß axis and expression of iNOS. Overall, these findings suggest that Drp1-dependent mitochondrial fission induces NF-κB nuclear translocation and NLRP3 inflammasome activation that may further contribute to olfactory bulb disturbances.

The expression of glucose transporters and mitochondrial division and fusion proteins in rats exposed to hypoxic preconditioning to attenuate propofol neurotoxicity.

Purpose: Evidence has shown that propofol may cause widespread apoptotic neurodegeneration. Hypoxic preconditioning has been demonstrated to provide neuroprotection and brain recovery from both acute and chronic neurodegeneration in several cellular and animal models. However, the mechanism has not been well elucidated. Therefore, the present study was designed to investigate the expression of glucose transporters (GLUT1 and GLUT3) and mitochondrial division and fusion (Drp1 and Mfn2) proteins in rats exposed to hypoxic preconditioning to attenuate propofol neurotoxicity.Methods: Propofol (100 mg/kg) was given to 7-day-old Sprague-Dawley rats; in some rats, hypoxic preconditioning was administered before intraperitoneal propofol injection by subjecting rats to five cycles of 10 min of hypoxia (8% O2) and 10 min of normoxia (21% O2). Then, the rats were allowed to breathe room air for 2 h. Neuronal mitochondrial morphology was observed by transmission electron microscopy. ATP content was detected using an ATP assay kit. The expression levels of GLUT1, GLUT3, pDrp1, Drp1 and Mfn2 were detected by Western blot, and the expression levels of GLUT1 and GLUT3 were further examined by immunohistochemistry.Results: Propofol damaged mitochondria, and decreased ATP content and GLUT3 and pDrp1 protein expression. However, our results suggested that hypoxic preconditioning could attenuate propofol neurotoxicity by reducing mitochondrial damage and increasing ATP content and pDrp1, GLUT1 and GLUT3 protein expression.Conclusion: Hypoxic preconditioning reduced propofol-induced damage in the hippocampus of neonatal rats by attenuating the increase in mitochondrial division and decrease in GLUT3 expression.

Dopamine D1 receptor agonism induces dynamin related protein-1 inhibition to improve mitochondrial biogenesis and dopaminergic neurogenesis in rat model of Parkinson's disease.

Dopamine (DA) neurotransmitter act on dopamine receptors (D1-D5) to regulate motor functions, reward, addiction and cognitive behavior. The depletion of DA in midbrain due to degeneration of nigral dopaminergic (DAergic) neurons leads to Parkinson's disease (PD). DA agonist and levodopa (L-DOPA) are the only therapies used for symptomatic relief in PD. However, the role of DA receptors in PD pathogenesis and how they are associated with mitochondrial functions and DAergic neurogenesis is still not known. Here, we investigated the mechanistic aspect of DA D1 receptor mediated control of DAergic neurogenesis, motor behavior and mitochondrial functions in rat PD model. The pharmacological activation of D1 receptors markedly improved motor deficits, mitochondrial biogenesis, ATP levels, mitochondrial membrane potential and defended nigral DAergic neurons against 6-hydroxydopamine (6-OHDA) induced neurotoxicity in adult rats. However, the D1 agonist mediated effects were abolished following D1 receptor antagonist treatment in 6-OHDA lesioned rats. Interestingly, pharmacological inhibition of dynamin related protein-1 (Drp-1) by Mdivi-1 in D1 antagonist treated PD rats, significantly restored behavioral deficits, mitochondrial functions, mitochondrial biogenesis and increased the number of newborn DAergic neurons in substantia nigra pars compacta (SNpc). Drp-1 inhibition mediated neuroprotective effects in PD rats were associated with increased level of protein kinase-B/Akt and extracellular-signal-regulated kinase (ERK). Taken together, our data suggests that dopamine D1 receptor mediated reduction in mitochondrial fission and enhanced DAergic neurogenesis may involve Drp-1 inhibition which led to improved behavioral recovery in PD rats.

Photochemical tissue bonding promotes the proliferation and migration of injured tenocytes through ROS/RhoA/NF-κB/Dynamin 2 signaling pathway.

Photochemical tissue bonding (PTB) has been found to promote the healing of Achilles tendon tissue injury and to reduce postoperative complications. However, the underlying cellular and molecular mechanisms are not clear. In this study, the cell proliferation, ROS generation, migration and the protein expression of DNM2, NF-κB p65, TGF-ß1 and VEGF in tenocytes after PTB treatment were measured by CCK-8, flow cytometry, Transwell and western blot assay, respectively. And those in tenocytes after DNM2 silencing or overexpressing or treatment with inhibitors of NF-κB, ROS and RhoA were also measured. Our results showed that 10 mW PTB treatment for 80 and 120 s significantly increased cell proliferation and increased ROS generation in tenocytes. 10 mW PTB treatment for 40 and 80 s significantly activated RhoA and increased the protein expression of DNM2, NF-κB p65, TGF-ß1 and VEGF, but 10 mW PTB treatment for 120 s decreased the protein expression of those. DNM2 silencing significantly suppressed cell migration and the expression of DNM2, TGF-ß1, and VEGF in tenocytes after PTB treatment (10 mW, 80 s), which was inhibited by DNM2 overexpression. Individual treatment with inhibitor of NF-κB, ROS, and RhoA in tenocytes showed decreased protein expression of DNM2, TGF-ß1, and VEGF. Moreover, in vivo experiment found that PTB treatment significantly inhibited cell apoptosis and the expression of DNM2, NF-κB p65, RhoA, TGF-ß1, and VEGF in a time-dependent manner. Taken together, our results suggest that PTB promotes the proliferation and migration of injured tenocytes through ROS/RhoA/NF-κB/DNM2 signaling pathway.

Iron overload triggers mitochondrial fragmentation via calcineurin-sensitive signals in HT-22 hippocampal neuron cells.

The accumulation of iron in neurons has been proposed to contribute to the pathology of numerous neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. However, insufficient research has been conducted on the precise mechanism underlying iron toxicity in neurons. In this study, we investigated mitochondrial dynamics in hippocampal HT-22 neurons exposed to ferric ammonium citrate (FAC) as a model of iron overload and neurodegeneration. Incubation with 150 µM FAC for 48 h resulted in decreased cell viability and apoptotic death in HT-22 cells. The FAC-induced iron overload triggered mitochondrial fragmentation, which was accompanied by Drp1(Ser637) dephosphorylation. Iron chelation with deferoxamine prevented the FAC-induced mitochondrial fragmentation and apoptotic cell death by inhibiting Drp1(Ser637) dephosphorylation. In addition, a S637D mutation of Drp1, which resulted in a phosphorylation-mimetic form of Drp1 at Ser637, protected against the FAC-induced mitochondrial fragmentation and neuronal apoptosis. FK506 and cyclosporine A, inhibitors of calcineurin activation, determined that calcineurin was associated with the iron-induced changes in mitochondrial morphology and the phosphorylation levels of Drp1. These results indicate that the FAC-induced dephosphorylation of Drp1-dependent mitochondrial fragmentation was rescued by the inhibition of calcineurin activation. Therefore, these findings suggest that calcineurin-mediated phosphorylation of Drp1(Ser637) acts as a key regulator of neuronal cell loss by modulating mitochondrial dynamics in iron-induced toxicity. These results may contribute to the development of novel therapies for treatment of neurodegenerative disorders related to iron toxicity.

Pharmacological targeting of actin-dependent dynamin oligomerization ameliorates chronic kidney disease in diverse animal models.

Dysregulation of the actin cytoskeleton in podocytes represents a common pathway in the pathogenesis of proteinuria across a spectrum of chronic kidney diseases (CKD). The GTPase dynamin has been implicated in the maintenance of cellular architecture in podocytes through its direct interaction with actin. Furthermore, the propensity of dynamin to oligomerize into higher-order structures in an actin-dependent manner and to cross-link actin microfilaments into higher-order structures has been correlated with increased actin polymerization and global organization of the actin cytoskeleton in the cell. We found that use of the small molecule Bis-T-23, which promotes actin-dependent dynamin oligomerization and thus increased actin polymerization in injured podocytes, was sufficient to improve renal health in diverse models of both transient kidney disease and CKD. In particular, administration of Bis-T-23 in these renal disease models restored the normal ultrastructure of podocyte foot processes, lowered proteinuria, lowered collagen IV deposits in the mesangial matrix, diminished mesangial matrix expansion and extended lifespan. These results further establish that alterations in the actin cytoskeleton of kidney podocytes is a common hallmark of CKD, while also underscoring the substantial regenerative potential of injured glomeruli and identifying the oligomerization cycle of dynamin as an attractive potential therapeutic target to treat CKD.

Lysine acetylation induced by chronic ethanol consumption impairs dynamin-mediated clathrin-coated vesicle release.

UNLABELLED: The liver is the major site of ethanol metabolism and thus sustains the most injury from chronic alcohol consumption. Ethanol metabolism by the hepatocyte leads to the generation of reactive metabolites and oxygen radicals that can readily adduct DNA, lipids, and proteins. More recently, it has become apparent that ethanol consumption also leads to increased post-translational modifications of the natural repertoire, including lysine hyperacetylation. Previously, we determined that alcohol consumption selectively impairs clathrin-mediated internalization in polarized hepatocytes. However, neither the step at which the block occurs nor the mechanism responsible for the defect have been identified. To identify the specific step at which clathrin-mediated internalization is impaired, we examined the distributions, levels, and assembly of selected components of the clathrin machinery in control and ethanol-treated cells. To determine whether the impairment is caused by ethanol-induced lysine acetylation, we also examined the same coat components in cells treated with trichostatin A (TSA), a deacetylase inhibitor that leads to protein hyperacetylation in the absence of ethanol. CONCLUSION: We determined that both ethanol and TSA impair internalization at a late stage before vesicle fission. We further determined that this defect is likely the result of decreased dynamin recruitment to the necks of clathrin-coated invaginations resulting in impaired vesicle budding. These results also raise the exciting possibility that agents that promote lysine deacetylation may be effective therapeutics for the treatment of alcoholic liver disease.

On the mechanism of internalization of alpha-synuclein into microglia: roles of ganglioside GM1 and lipid raft.

ALpha-synuclein (alpha-syn) has been known to be a key player of the pathogenesis of Parkinson's disease and has recently been detected in extracellular biological fluids and shown to be rapidly secreted from cells. The penetration of alpha-syn into cells has also been observed. In this study, we observed that dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol, a glucosyltransferase inhibitor, and proteinase K inhibited the internalization of extracellular monomeric alpha-syn into BV-2 cells, and the addition of monosialoganglioside GM1 ameliorated the inhibition of alpha-syn internalization in dl-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol-treated BV-2 cells. Furthermore, inhibition of clathrin-, caveolae-, and dynamin-dependent endocytosis did not prevent the internalization of alpha-syn, but disruption of lipid raft inhibited it. Inhibition of macropinocytosis and disruption of actin and microtubule structures also did not inhibit the internalization of alpha-syn. In addition, we further confirmed these observations by co-culture system of BV-2 cells and alpha-syn-over-expressing SH-SY5Y cells. These findings suggest that extracellular alpha-syn is internalized into microglia via GM1 as well as hitherto-unknown protein receptors in clathrin-, caveolae-, and dynamin-independent, but lipid raft-dependent manner. Elucidation of the mechanism involved in internalization of alpha-syn should be greatly helpful in the development of new treatments of alpha-syn-related neurodegenerative diseases.

A clathrin/dynamin- and mannose-6-phosphate receptor-independent pathway for granzyme B-induced cell death.

The 280-kD cation-independent mannose-6-phosphate receptor (MPR) has been shown to play a role in endocytic uptake of granzyme B, since target cells overexpressing MPR have an increased sensitivity to granzyme B-mediated apoptosis. On this basis, it has been proposed that cells lacking MPR are poor targets for cytotoxic lymphocytes that mediate allograft rejection or tumor immune surveillance. In the present study, we report that the uptake of granzyme B into target cells is independent of MPR. We used HeLa cells overexpressing a dominant-negative mutated (K44A) form of dynamin and mouse fibroblasts overexpressing or lacking MPR to show that the MPR/clathrin/dynamin pathway is not required for granzyme B uptake. Consistent with this observation, cells lacking the MPR/clathrin pathway remained sensitive to granzyme B. Exposure of K44A-dynamin-overexpressing and wild-type HeLa cells to granzyme B with sublytic perforin resulted in similar apoptosis in the two cell populations, both in short and long term assays. Granzyme B uptake into MPR-overexpressing L cells was more rapid than into MPR-null L cells, but the receptor-deficient cells took up granzyme B through fluid phase micropinocytosis and remained sensitive to it. Contrary to previous findings, we also demonstrated that mouse tumor allografts that lack MPR expression were rejected as rapidly as tumors that overexpress MPR. Entry of granzyme B into target cells and its intracellular trafficking to induce target cell death in the presence of perforin are therefore not critically dependent on MPR or clathrin/dynamin-dependent endocytosis.