Mitophagy activation by rapamycin enhances mitochondrial function and cognition in 5×FAD mice.

Alzheimer's disease (AD) is the most prevalent form of dementia, characterized by severe mitochondrial dysfunction, which is an intracellular process that is significantly compromised in the early stages of AD. Mitophagy, the selective removal of damaged mitochondria, is a potential therapeutic strategy for AD. Rapamycin, a mammalian target of rapamycin (mTOR) inhibitor, augmented autophagy and mitigated cognitive impairment. Our study revealed that rapamycin enhances cognitive function by activating mitophagy, alleviating neuronal loss, and improving mitochondrial dysfunction in 5 ×FAD mice. Interestingly, the neuroprotective effect of rapamycin in AD were negated by treatment with 3-MA, a mitophagy inhibitor. Overall, our findings suggest that rapamycin ameliorates cognitive impairment in 5 ×FAD mice via mitophagy activation and its downstream PINK1-Parkin pathway, which aids in the clearance of amyloid-ß (Aß) and damaged mitochondria. This study reveals a novel mechanism involving mitophagy regulation underlying the therapeutic effect of rapamycin in AD. This study provides new insights and therapeutic targets for rapamycin in the treatment of AD. However, there are still some shortcomings in this topic; if we can further knock out the PINK1/Parkin gene in animals or use siRNA technology, we can further confirm the experimental results.

Activation of mitophagy improves cognitive dysfunction in diabetic mice with recurrent non-severe hypoglycemia.

Recurrent non-severe hypoglycemia (RH) in patients with diabetes might be associated with cognitive impairment. Previously, we found that mitochondrial dysfunction plays an important role in this pathological process; however, the mechanism remains unclear. The objective of this study was to determine the molecular mechanisms of mitochondrial damage associated with RH in diabetes mellitus (DM). We found that RH is associated with reduced hippocampal mitophagy in diabetic mice, mainly manifested by reduced autophagosome formation and impaired recognition of impaired mitochondria, mediated by the PINK1/Parkin pathway. The same impaired mitophagy initiation was observed in an in vitro high-glucose cultured astrocyte model with recurrent low-glucose interventions. Promoting autophagosome formation and activating PINK1/Parkin-mediated mitophagy protected mitochondrial function and cognitive function in mice. The results showed that impaired mitophagy is involved in the occurrence of mitochondrial dysfunction, mediating the neurological impairment associated with recurrent low glucose under high glucose conditions.

Morroniside induces cardiomyocyte cell cycle activity and promotes cardiac repair after myocardial infarction in adult rats.

Introduction: Acute myocardial infarction (AMI) is characterized by the loss of cardiomyocytes, which impairs cardiac function and eventually leads to heart failure. The induction of cardiomyocyte cell cycle activity provides a new treatment strategy for the repair of heart damage. Our previous study demonstrated that morroniside exerts cardioprotective effects. This study investigated the effects and underlying mechanisms of action of morroniside on cardiomyocyte cell cycle activity and cardiac repair following AMI. Methods: Neonatal rat cardiomyocytes (NRCMs) were isolated and exposed to oxygen-glucose deprivation (OGD) in vitro. A rat model of AMI was established by ligation of the left anterior descending coronary artery (LAD) in vivo. Immunofluorescence staining was performed to detect newly generated cardiomyocytes. Western blotting was performed to assess the expression of cell cycle-related proteins. Electrocardiography (ECG) was used to examine pathological Q waves. Masson's trichrome and wheat germ agglutinin (WGA) staining assessed myocardial fibrosis and hypertrophy. Results: The results showed that morroniside induced cardiomyocyte cell cycle activity and increased the levels of cell cycle proteins, including cyclin D1, CDK4, cyclin A2, and cyclin B1, both in vitro and in vivo. Moreover, morroniside reduced myocardial fibrosis and remodeling. Discussion: In conclusion, our study demonstrated that morroniside stimulates cardiomyocyte cell cycle activity and cardiac repair in adult rats, and that these effects may be related to the upregulation of cell cycle proteins.

Morroniside Regulates Endothelial Cell Function via the EphrinB Signaling Pathway after Oxygen-Glucose Deprivation In Vitro.

Proangiogenic treatment is a potential treatment for acute myocardial infarction (AMI). Morroniside was previously discovered to increase post-AMI angiogenesis in rats as well as the proliferation of rat coronary artery endothelial cells (RCAECs). However, the effects of morroniside on other endothelial cell (EC) functions and underlying mechanisms are unknown. To further clarify the vascular biological activity of morroniside, this work focused on investigating how morroniside influenced endothelial cell functions, such as cell viability, tube formation capacity, migration, and adhesion, and to explore the signaling pathway. Oxygen-glucose deprivation causes ischemic damage in RCAECs (OGD). In vitro investigations were carried out to explore the involvement of morroniside in EC function and pathways mediated by ephrinB. The results revealed that the number of BrdU+ cells and cell viability in the high-dose group were considerably greater than in the OGD group (P < 0.05). The ability of tube formation evaluated by total tube length, tube-like structural junction, and tube area was significantly higher in the morroniside group than in the OGD group (P < 0.001). Morroniside considerably improved migration and adhesion abilities compared to OGD group (P < 0.05, P < 0.01, P < 0.001). The protein expression levels of the ephrinB reverse signaling pathway were substantially greater in the morroniside group than in the OGD group (P < 0.05, P < 0.01). In conclusion, the current study demonstrated that morroniside modulates endothelial cell function via ephrinB reverse signaling pathways and provided a novel insight and therapeutic strategy into vascular biology.

SIRT3 alleviates mitochondrial dysfunction induced by recurrent low glucose and improves the supportive function of astrocytes to neurons.

Hypoglycemia is an independent risk factor of cognitive impairment in patients with diabetes. Our previous study indicated that dysfunction of astrocytic mitochondria induced by recurrent low glucose (RLG) may account for hypoglycemia-associated neuronal injury and cognitive decline. Sirtuin 3 (SIRT3) is a key deacetylase for mitochondrial proteins and has recently been demonstrated to be an important regulator of mitochondrial function. However, whether mitochondrial dysfunction due to hypoglycemia is associated with astrocytic SIRT3 remains unclear, and few studies have focused on the impact of astrocytic SIRT3 on neuronal survival. In the present work, primary mouse cortical astrocytes cultured in normal glucose (5.5 mM) and high glucose (16.5 mM) were treated with five rounds of RLG (0.1 mM). The results showed that RLG suppressed SIRT3 expression in a glucose-dependent manner. High-glucose culture considerably increased the vulnerability of SIRT3 to RLG, leading to disrupted mitochondrial morphology in astrocytes. Overexpression of SIRT3 markedly improved astrocytic mitochondrial function and reduced RLG-induced oxidative stress. Moreover, SIRT3 suppressed a shift towards a neuroinflammatory A1-like reactive phenotype of astrocytes in response to RLG with reduced IL-1ß, IL-6, and TNFα levels. Furthermore, it elevated brain-derived neurotrophic factor (BDNF) levels and promoted neurite growth by activating BDNF/TrkB signaling in the co-cultured neurons. The present study reveals the probable crosstalk between neurons and astrocytes after hypoglycemic exposure and provides a potential target in treating hypoglycemia-associated neuronal injury.

GLP-1 improves the neuronal supportive ability of astrocytes in Alzheimer's disease by regulating mitochondrial dysfunction via the cAMP/PKA pathway.

The glucagon-like peptide-1 (GLP-1) was shown to have neuroprotective effects in Alzheimer's disease (AD). However, the underlying mechanism remains elusive. Astrocytic mitochondrial abnormalities have been revealed to constitute important pathologies. In the present study, we investigated the role of astrocytic mitochondria in the neuroprotective effect of GLP-1 in AD. To this end, 6-month-old 5 × FAD mice were subcutaneously treated with liraglutide, a GLP-1 analogue (25 nmol/kg/qd) for 8 weeks. Liraglutide ameliorated mitochondrial dysfunction and prevented neuronal loss with activation of the cyclic adenosine 3',5'-monophosphate (cAMP)/phosphorylate protein kinase A (PKA) pathway in the brain of 5 × FAD mice. Next, we exposed astrocytes to ß-amyloid (Aß) in vitro and treated them with GLP-1. By activating the cAMP/PKA pathway, GLP-1 increased the phosphorylation of DRP-1 at the s637 site and mitigated mitochondrial fragmentation in Aß-treated astrocytes. GLP-1 further improved the Aß-induced energy failure, mitochondrial reactive oxygen species (ROS) overproduction, mitochondrial membrane potential (MMP) collapse, and cell toxicity in astrocytes. Moreover, GLP-1 also promoted the neuronal supportive ability of Aß-treated astrocytes via the cAMP/PKA pathway. This study revealed a new mechanism behind the neuroprotective effect of GLP-1 in AD.

Ubiquitin C-terminal hydrolase-L1 as a biomarker for ischemic and traumatic brain injury in rats.

Ubiquitin C-terminal hydrolase-L1 (UCH-L1), also called neuronal-specific protein gene product 9.5, is a highly abundant protein in the neuronal cell body and has been identified as a possible biomarker on the basis of a recent proteomic study. In this study, we examined whether UCH-L1 was significantly elevated in cerebrospinal fluid (CSF) following controlled cortical impact (CCI) and middle cerebral artery occlusion (MCAO; model of ischemic stroke) in rats. Quantitative immunoblots of rat CSF revealed a dramatic elevation of UCH-L1 protein 48 h after severe CCI and as early as 6 h after mild (30 min) and severe (2 h) MCAO. A sandwich enzyme-linked immunosorbent assay constructed to measure UCH-L1 sensitively and quantitatively showed that CSF UCH-L1 levels were significantly elevated as early as 2 h and up to 48 h after CCI. Similarly, UCH-L1 levels were also significantly elevated in CSF from 6 to 72 h after 30 min of MCAO and from 6 to 120 h after 2 h of MCAO. These data are comparable to the profile of the calpain-produced alphaII-spectrin breakdown product of 145 kDa biomarker. Importantly, serum UCH-L1 biomarker levels were also significantly elevated after CCI. Similarly, serum UCH-L1 levels in the 2-h MCAO group were significantly higher than those in the 30-min group. Taken together, these data from two rat models of acute brain injury strongly suggest that UCH-L1 is a candidate brain injury biomarker detectable in biofluid compartments (CSF and serum).

Ubiquitin C-terminal hydrolase is a novel biomarker in humans for severe traumatic brain injury.

OBJECTIVE: Ubiquitin C-terminal hydrolase (UCH-L1), also called neuronal-specific protein gene product (PGP 9.3), is highly abundant in neurons. To assess the reliability of UCH-L1 as a potential biomarker for traumatic brain injury (TBI) this study compared cerebrospinal fluid (CSF) levels of UCH-L1 from adult patients with severe TBI to uninjured controls; and examined the relationship between levels with severity of injury, complications and functional outcome. DESIGN: This study was designed as prospective case control study. PATIENTS: This study enrolled 66 patients, 41 with severe TBI, defined by a Glasgow coma scale (GCS) score of < or =8, who underwent intraventricular intracranial pressure monitoring and 25 controls without TBI requiring CSF drainage for other medical reasons. SETTING: : Two hospital system level I trauma centers. MEASUREMENTS AND MAIN RESULTS: Ventricular CSF was sampled from each patient at 6, 12, 24, 48, 72, 96, 120, 144, and 168 hrs following TBI and analyzed for UCH-L1. Injury severity was assessed by the GCS score, Marshall Classification on computed tomography and a complicated postinjury course. Mortality was assessed at 6 wks and long-term outcome was assessed using the Glasgow outcome score 6 months after injury. TBI patients had significantly elevated CSF levels of UCH-L1 at each time point after injury compared to uninjured controls. Overall mean levels of UCH-L1 in TBI patients was 44.2 ng/mL (+/-7.9) compared with 2.7 ng/mL (+/-0.7) in controls (p <.001). There were significantly higher levels of UCH-L1 in patients with a lower GCS score at 24 hrs, in those with postinjury complications, in those with 6-wk mortality, and in those with a poor 6-month dichotomized Glasgow outcome score. CONCLUSIONS: These data suggest that this novel biomarker has the potential to determine injury severity in TBI patients. Further studies are needed to validate these findings in a larger sample.

Acute NMDA toxicity in cultured rat cerebellar granule neurons is accompanied by autophagy induction and late onset autophagic cell death phenotype.

BACKGROUND: Autophagy, an intracellular response to stress, is characterized by double membrane cytosolic vesicles called autophagosomes. Prolonged autophagy is known to result in autophagic (Type II) cell death. This study examined the potential role of an autophagic response in cultured cerebellar granule neurons challenged with excitotoxin N-methyl-D-aspartate (NMDA). RESULTS: NMDA exposure induced light chain-3 (LC-3)-immunopositive and monodansylcadaverine (MDC) fluorescent dye-labeled autophagosome formation in both cell bodies and neurites as early as 3 hours post-treatment. Elevated levels of Beclin-1 and the autophagosome-targeting LC3-II were also observed following NMDA exposure. Prolonged exposure of the cultures to NMDA (8-24 h) generated MDC-, LC3-positive autophagosomal bodies, concomitant with the neurodegenerative phase of NMDA challenge. Lysosomal inhibition studies also suggest that NMDA-treatment diverted the autophagosome-associated LC3-II from the normal lysosomal degradation pathway. Autophagy inhibitor 3-methyladenine significantly reduced NMDA-induced LC3-II/LC3-I ratio increase, accumulation of autophagosomes, and suppressed NMDA-mediated neuronal death. ATG7 siRNA studies also showed neuroprotective effects following NMDA treatment. CONCLUSIONS: Collectively, this study shows that autophagy machinery is robustly induced in cultured neurons subjected to prolonged exposure to excitotoxin, while autophagosome clearance by lysosomal pathway might be impaired. Our data further show that prolonged autophagy contributes to cell death in NMDA-mediated excitotoxicity.

Electrical stimulation of the lateral cerebellar nucleus promotes neurogenesis in rats after motor cortical ischemia.

Deep brain stimulation (DBS) has been tentatively explored to promote motor recovery after stroke. Stroke could transiently activate endogenous self-repair processes, including neurogenesis in the subventricular zone (SVZ). In this regard, it is of considerable clinical interest to study whether DBS of the lateral cerebellar nucleus (LCN) could promote neurogenesis in the SVZ for functional recovery after stroke. In the present study, rats were trained on the pasta matrix reaching task and the ladder rung walking task before surgery. And then an electrode was implanted in the LCN following cortical ischemia induced by endothelin-1 injection. After 1 week of recovery, LCN DBS coupled with motor training for two weeks promoted motor function recovery, and reduced the infarct volumes post-ischemia. LCN DBS augmented poststroke neurogenetic responses, characterized by proliferation of neural progenitor cells (NPCs) and neuroblasts in the SVZ and subsequent differentiation into neurons in the ischemic penumbra at 21 days poststroke. DBS with the same stimulus parameters at 1 month after ischemia could also increase nascent neuroblasts in the SVZ and newly matured neurons in the perilesional cortex at 42 days poststroke. These results suggest that LCN DBS promotes endogenous neurogenesis for neurorestoration after cortical ischemia.

Morroniside enhances angiogenesis and improves cardiac function following acute myocardial infarction in rats.

Angiogenesis is critical for re-establishing blood supply to the ischemic myocardium after acute myocardial infarction (AMI). This study aimed to investigate the effects of morroniside on angiogenesis after AMI and explored associated proangiogenic mechanisms. A rat model of AMI was established by ligation of the left anterior descending coronary artery followed by administration of three doses of morroniside. Immunofluorescence staining was performed to identify newly generated endothelial cells and arterioles. The protein expression levels associated with angiogenesis were examined by western blots. Echocardiography was used to examine cardiac function. Our data revealed that morroniside promoted angiogenesis and improved cardiac function in rats with AMI. The proangiogenic effect of morroniside might be mediated by the VEGFA/VEGF receptor 2 signaling pathway.

Multiple alphaII-spectrin breakdown products distinguish calpain and caspase dominated necrotic and apoptotic cell death pathways.

Apoptosis and oncotic necrosis in neuronal and glial cells have been documented in many neurological diseases. Distinguishing between these two major types of cell death in different neurological diseases is needed in order to better reveal the injury mechanisms so as to open up opportunities for therapy development. Accumulating evidence suggests apoptosis and oncosis epitomize the extreme ends of a broad spectrum of morphological and biochemical events. Biochemical markers that can distinguish between the calpain and caspase dominated types of cell death would help in this process. In this study, three chemical agents, maitotoxin (MTX), staurosporine (STS) and thylenediaminetetraacetic acid (EDTA), were used to induce different types of cell death in PC12 neuronal-like cells. MTX-induced necrosis, as determined by the increased levels of calpain-specific cleaved fragments of spectrin by antibodies specific to the calpain-cleaved 150 kDa alphaII-spectrin breakdown product (SBDP150) and 145 kDa alphaII-spectrin breakdown product (SBDP145). In this paradigm, there were no detectable SBDP150i and SBDP120 fragments as determined by antibodies specific to the caspase-cleaved specific fragments similar to those seen in the EDTA-mediated apoptotic PC-12 cells. In contrast to the calpain specific MTX necrosis treatment and the caspase EDTA apoptotic treatment is the STS treatment which induced both proteases as shown by the increase in all the SBDP fragments. Furthermore, compared to SBDP150, SBDP145 appears to be a more specific and sensitive biomarker for calpain activation. Taken together, our results suggested calpains and caspases which dominate the two major types of cell death could be independently discriminated by specifically examining the multiple alphaII-spectrin cleavage breakdown products.

Comparing calpain- and caspase-3-mediated degradation patterns in traumatic brain injury by differential proteome analysis.

A major theme of TBI (traumatic brain injury) pathology is the over-activation of multiple proteases. We have previously shown that calpain-1 and -2, and caspase-3 simultaneously produced alphaII-spectrin BDPs (breakdown products) following TBI. In the present study, we attempted to identify a comprehensive set of protease substrates (degradome) for calpains and caspase-3. We further hypothesized that the TBI differential proteome is likely to overlap significantly with the calpain- and caspase-3-degradomes. Using a novel HTPI (high throughput immunoblotting) approach and 1000 monoclonal antibodies (PowerBlottrade mark), we compared rat hippocampal lysates from 4 treatment groups: (i) naïve, (ii) TBI (48 h after controlled cortical impact), (iii) in vitro calpain-2 digestion and (iv) in vitro caspase-3 digestion. In total, we identified 54 and 38 proteins that were vulnerable to calpain-2 and caspase-3 proteolysis respectively. In addition, the expression of 48 proteins was down-regulated following TBI, whereas that of only 9 was up-regulated. Among the proteins down-regulated in TBI, 42 of them overlapped with the calpain-2 and/or caspase-3 degradomes, suggesting that they might be proteolytic targets after TBI. We further confirmed several novel TBI-linked proteolytic substrates, including betaII-spectrin, striatin, synaptotagmin-1, synaptojanin-1 and NSF (N-ethylmaleimide-sensitive fusion protein) by traditional immunoblotting. In summary, we demonstrated that HTPI is a novel and powerful method for studying proteolytic pathways in vivo and in vitro.

Morroniside promotes angiogenesis and further improves microvascular circulation after focal cerebral ischemia/reperfusion.

Preservation of cerebral microvascular functional integrity is crucial for protecting and repairing the brain after stroke. Our previous study demonstrated that morroniside promoted angiogenesis 7days after stroke. The current study aimed to further evaluate the long-term effects of morroniside on angiogenesis and to examine whether angiogenesis induced by morroniside could improve blood flow velocity. Sprague-Dawley rats were subjected to middle cerebral artery occlusion (MCAO), and morroniside was then administered once per day at a dose of 270mg/kg. New vessel formation and the expression of ephrinB2/VEGFR2 signaling pathway components were examined 14days after MCAO to examine angiogenesis and the associated mechanisms. The dynamics of regional cerebral blood flow (rCBF) and the number of vessels of the leptomeningeal anastomoses were analyzed to characterize microvascular circulation 3days after MCAO. We demonstrated that morroniside promoted angiogenesis by regulating the ephrinB2/VEGFR2 signaling pathway 14days post-ischemia. By 3days post-ischemia, morroniside improved rCBF and increased the number of vessels of the leptomeningeal anastomoses. Moreover, morroniside decreased the infarct volume and improved neurological function 14days after MCAO. Our findings suggest that morroniside promoted long-term angiogenesis, thereby improving microvascular circulation and neurological function. It suggested that the angiogenic mechanism of morroniside might be mediated by the ephrinB2/VEGFR2 signaling pathway.

Degradation of ßII-Spectrin Protein by Calpain-2 and Caspase-3 Under Neurotoxic and Traumatic Brain Injury Conditions.

A major consequence of traumatic brain injury (TBI) is the rapid proteolytic degradation of structural cytoskeletal proteins. This process is largely reflected by the interruption of axonal transport as a result of extensive axonal injury leading to neuronal cell injury. Previous work from our group has described the extensive degradation of the axonally enriched cytoskeletal αII-spectrin protein which results in molecular signature breakdown products (BDPs) indicative of injury mechanisms and to specific protease activation both in vitro and in vivo. In the current study, we investigated the integrity of ßII-spectrin protein and its proteolytic profile both in primary rat cerebrocortical cell culture under apoptotic, necrotic, and excitotoxic challenge and extended to in vivo rat model of experimental TBI (controlled cortical impact model). Interestingly, our results revealed that the intact 260-kDa ßII-spectrin is degraded into major fragments (ßII-spectrin breakdown products (ßsBDPs)) of 110, 108, 85, and 80 kDa in rat brain (hippocampus and cortex) 48 h post-injury. These ßsBDP profiles were further characterized and compared to an in vitro ßII-spectrin fragmentation pattern of naive rat cortex lysate digested by calpain-2 and caspase-3. Results revealed that ßII-spectrin was degraded into major fragments of 110/85 kDa by calpain-2 activation and 108/80 kDa by caspase-3 activation. These data strongly support the hypothesis that in vivo activation of multiple protease system induces structural protein proteolysis involving ßII-spectrin proteolysis via a specific calpain and/or caspase-mediated pathway resulting in a signature, protease-specific ßsBDPs that are dependent upon the type of neural injury mechanism. This work extends on previous published work that discusses the interplay spectrin family (αII-spectrin and ßII-spectrin) and their susceptibility to protease proteolysis and their implication to neuronal cell death mechanisms.

Dual vulnerability of tau to calpains and caspase-3 proteolysis under neurotoxic and neurodegenerative conditions.

Axonally specific microtubule-associated protein tau is an important component of neurofibrillary tangles found in AD (Alzheimer's disease) and other tauopathy diseases such as CTE (chronic traumatic encephalopathy). Such tau aggregate is found to be hyperphosphorylated and often proteolytically fragmented. Similarly, tau is degraded following TBI (traumatic brain injury). In the present study, we examined the dual vulnerability of tau to calpain and caspase-3 under neurotoxic and neurodegenerative conditions. We first identified three novel calpain cleavage sites in rat tau (four-repeat isoform) as Ser130↓Lys131, Gly157↓Ala158 and Arg380↓Glu381. Fragment-specific antibodies to target the major calpain-mediated TauBDP-35K (35 kDa tau-breakdown product) and the caspase-mediated TauBDP-45K respectively were developed. In rat cerebrocortical cultures treated with excitotoxin [NMDA (N-methyl-D-aspartate)], tau is significantly degraded into multiple fragments, including a dominant signal of calpain-mediated TauBDP-35K with minimal caspase-mediated TauBDP-45K. Following apoptosis-inducing EDTA treatment, tau was truncated only to TauBDP-48K/45K-exclusively by caspase. Cultures treated with another apoptosis inducer STS (staurosporine), dual fragmentation by calpain (TauBDP-35K) and caspase-3 (TauBDP-45K) was observed. Tau was also fragmented in injured rat cortex following TBI in vivo to BDPs of 45-42 kDa (minor), 35 kDa and 15 kDa, followed by TauBDP-25K. Calpain-mediated TauBDP-35K-specific antibody confirmed robust signals in the injured cortex, while caspase-mediated TauBDP-45K-specific antibody only detected faint signals. Furthermore, intravenous administration of a calpain-specific inhibitor SNJ-1945 strongly suppressed the TauBDP-35K formation. Taken together, these results suggest that tau protein is dually vulnerable to calpain and caspase-3 proteolysis under different neurotoxic and injury conditions.

Calpain- and caspase-mediated alphaII-spectrin and tau proteolysis in rat cerebrocortical neuronal cultures after ecstasy or methamphetamine exposure.

Abuse of 3,4-methylenedioxymethamphetamine (MDMA or Ecstasy) and methamphetamine (Meth or Speed) is a growing international problem with an estimated 250 million users of psychoactive drugs worldwide. It is important to demonstrate and understand the mechanism of neurotoxicity so potential prevention and treatment therapies can be designed. In this study rat primary cerebrocortical neuron cultures were challenged with MDMA and Meth (1 or 2 mM) for 24 and 48 h and compared to the excitotoxin N-methyl-D-aspartate (NMDA). The neurotoxicity of these drugs, as assessed by microscopy, lactate dehydrogenase release and immunoblot, was shown to be both dose- and time-dependent. Immunoblot analysis using biomarkers of cell death showed significant proteolysis of both alphaII-spectrin and tau proteins. Breakdown products of alphaII-spectrin (SBDPs) of 150, 145, and 120 kDa and tau breakdown products (TBDPs) of 45, 32, 26, and 14 kDa were observed. The use of the protease inhibitors calpain inhibitor SJA6017 and caspase inhibitors z-VAD-fmk and Z-D-DCB, attenuated drug-induced alphaII-spectrin and tau proteolysis. The calpain inhibitor reduced the calpain-induced breakdown products SBDP145 and TBDP14, but there was an offset increase in the caspase-mediated breakdown products SBDP120 and TBDP45. The caspase inhibitors, on the other hand, decreased SBDP120 and TBDP45. These data suggest that both MDMA and Meth trigger concerted proteolytic attacks of the structural proteins by both calpain and caspase family of proteases. The ability of the protease inhibitors to reduce the damage caused by these drugs suggests that the treatment arsenal could include similar drugs as possible tools to combat the drug-induced neurotoxicity in vivo.

Extensive degradation of myelin basic protein isoforms by calpain following traumatic brain injury.

Axonal injury is one of the key features of traumatic brain injury (TBI), yet little is known about the integrity of the myelin sheath. We report that the 21.5 and 18.5-kDa myelin basic protein (MBP) isoforms degrade into N-terminal fragments (of 10 and 8 kDa) in the ipsilateral hippocampus and cortex between 2 h and 3 days after controlled cortical impact (in a rat model of TBI), but exhibit no degradation contralaterally. Using N-terminal microsequencing and mass spectrometry, we identified a novel in vivo MBP cleavage site between Phe114 and Lys115. A MBP C-terminal fragment-specific antibody was then raised and shown to specifically detect MBP fragments in affected brain regions following TBI. In vitro naive brain lysate and purified MBP digestion showed that MBP is sensitive to calpain, producing the characteristic MBP fragments observed in TBI. We hypothesize that TBI-mediated axonal injury causes secondary structural damage to the adjacent myelin membrane, instigating MBP degradation. This could initiate myelin sheath instability and demyelination, which might further promote axonal vulnerability.