calpain and Mitochondrial-Diseases

Neurocognitive deficits seen in HIV-associated neurocognitive disorders (HANDs) are attributed to the release of soluble factors from CNS-resident, HIV-infected and/or activated macrophages and microglia. To study HIV-associated neurotoxicity, we used our in vitro model in which primary rat neuronal/glial cultures are treated with supernatants from cultured human monocyte-derived macrophages, infected with a CNS-isolated HIV-1 strain (HIV-MDM). We found that neuronal damage, detected as a loss of microtubule-associated protein-2 (MAP2), begins as early as 2h and is preceded by a loss of mitochondrial membrane potential (Δψ(m)). Interestingly, inhibitors of calpains, but not inhibitors of caspases, blocked MAP2 loss, however neither type of inhibitor prevented the loss of Δψ(m). To facilitate throughput for these studies, we refined a MAP2 cell-based-ELISA whose data closely compare with our standardized method of hand counting neurons. In addition, we developed a tetramethyl rhodamine methyl ester (TMRM)-based multi-well fluorescent plate assay for the evaluation of whole culture Δψ(m). Together, these findings indicate that calpain activation and loss of Δψ(m) may be parallel pathways to death in HIV-MDM-treated neurons and also demonstrate the validity of plate assays for assessing multiple experimental parameters as is useful for screening neurotherapeutics for neuronal damage and death.

Topics: AIDS Dementia Complex; Animals; Calpain; Cell Culture Techniques; Cell Death; Cells, Cultured; Macrophages; Membrane Potential, Mitochondrial; Microtubule-Associated Proteins; Mitochondrial Diseases; Nerve Degeneration; Neurons; Rats; Rats, Sprague-Dawley; Rhodamines

Calpain is a ubiquitous calcium-sensitive protease that is essential for normal physiologic neuronal function. However, mitochondrial-mediated-calcium homeostasis alterations may lead to its pathologic activation that jeopardizes neuronal structure and function. Here, we provide evidence to support a role for the involvement of calpain 1 in mitochondrial-induced neurodegeneration in a Parkinson's disease (PD) cellular model. We show that dysfunctional mitochondria increases cytosolic calcium, thereby, inducing calpain activation. Interestingly, its inhibition significantly attenuated the accumulation of alpha-synuclein oligomers and contributed to an increase of insoluble alpha-synuclein aggregates, known to be cytoprotective. Moreover, our data corroborate that calpain-1 overactivation in our mitochondrial-deficient cells promote caspase-3 activation. Overall, our findings further clarify the crucial role of dysfunctional mitochondria in the control of molecular mechanisms occurring in PD brain cells, providing a potentially novel correlation between the degradation of calpain substrates suggesting a putative role of calpain and calpain inhibition as a therapeutic tool in PD.

Topics: alpha-Synuclein; Calcium; Calcium Signaling; Calpain; Caspase 3; Cell Line, Transformed; Enzyme Activation; Humans; Inclusion Bodies; Mitochondria; Mitochondrial Diseases; Models, Biological; Nerve Degeneration; Parkinson Disease

Networks of neurons express persistent spontaneous network activity when maintained in dissociated cultures. Prolonged blockade of the spontaneous activity with tetrodotoxin (TTX) causes the eventual death of the neurons. In this study, we investigated some molecular mechanisms that may underlie the activity-suppressed slow degeneration of cortical neurons in culture. Already after 3-4 days of exposure to TTX, well before the neurons die, they began to express markers that lead to their eventual death, 7-10 days later. There was a reduction in glutamate receptor (GluR2) expression, a persistent increase in intracellular calcium concentration, activation of calpain, and an increase in spectrin breakdown products. At this point, blockade of GluR2-lacking GluR1 or calpain (either with a selective antagonist or through the natural regulator of calpain, calpastatin), protected cells from the toxic action of TTX. Subsequently, mitochondria lost their normal elongated shape as well as their membrane potential. Eventually, neurons activated caspase 3 and PUMA (p53 up-regulated modulator of apoptosis), hallmarks of neuronal apoptosis, and died. These experiments will lead to a better understanding of slow neuronal death, typical of neurodegenerative diseases.

Topics: Action Potentials; Animals; Animals, Newborn; Apoptosis; Calcium; Calcium Signaling; Calpain; Cells, Cultured; Cerebral Cortex; Energy Metabolism; Mitochondria; Mitochondrial Diseases; Nerve Degeneration; Nerve Net; Neural Pathways; Neurons; Rats; Rats, Wistar; Signal Transduction; Sodium Channel Blockers; Synaptic Transmission; Tetrodotoxin

Glutamate neurotoxicity is exacerbated when energy metabolism is impaired. In vitro studies show that neuronal death in these conditions is related to mitochondrial dysfunction, ATP depletion, and the loss of calcium homeostasis. We have recently observed that, in vivo, enhancement of glutamate toxicity elicited by previous mitochondrial inhibition does not involve severe ATP depletion, suggesting the involvement of other processes. Factors such as the activation of different proteases may determine the extent and type of cell death. Protease activation might be triggered by internal or external factors, such as mitochondrial damage or the activation of a particular glutamate receptor subtype. In the present study we aimed to investigate whether moderate inhibition of mitochondrial metabolism facilitates glutamate toxicity through caspase-3 or calpain activation, as well as the contribution of NMDA and non-NMDA glutamate ionotropic receptors to this activation. Rats were pre-treated with a subtoxic dose of 3-NP and 4 h later intrastriatally injected with glutamate. Results show that neither of these treatments alone (3-NP or Glu) or in combination (3-NP+Glu) activated caspase-3. Conversely, calpain activity is induced after glutamate injection both in intact and 3-NP pre-treated rats. Inhibition of calpain activity by MDL-28170 significantly prevented striatal damage. NMDA and non-NMDA receptors contributed equally to calpain activation and to the induction of neuronal death. Results suggest that enhancement of glutamate toxicity due to inhibition of mitochondrial metabolism in vivo, does not recruit caspase-dependent apoptosis but favors calpain activation through the stimulation of both subtypes of glutamate ionotropic receptors.

Topics: Animals; Brain Diseases, Metabolic; Calpain; Caspase 3; Cell Death; Enzyme Activation; Excitatory Amino Acid Antagonists; Glutamic Acid; Male; Mitochondria; Mitochondrial Diseases; Nerve Degeneration; Neurotoxins; Rats; Rats, Wistar; Receptors, Glutamate; Receptors, N-Methyl-D-Aspartate; Signal Transduction

Calpains, Ca(2+)-activated cysteine proteases, are cytosolic enzymes implicated in numerous cellular functions and pathologies. We identified a mitochondrial Ca(2+)-inducible protease that hydrolyzed a calpain substrate (SLLVY-AMC) and was inhibited by active site-directed calpain inhibitors as calpain 10, an atypical calpain lacking domain IV. Immunoblot analysis and activity assays revealed calpain 10 in the mitochondrial outer membrane, intermembrane space, inner membrane, and matrix fractions. Mitochondrial staining was observed when COOH-terminal green fluorescent protein-tagged calpain 10 was overexpressed in NIH-3T3 cells and the mitochondrial targeting sequence was localized to the NH(2)-terminal 15 amino acids. Overexpression of mitochondrial calpain 10 resulted in mitochondrial swelling and autophagy that was blocked by the mitochondrial permeability transition (MPT) inhibitor cyclosporine A. With the use of isolated mitochondria, Ca(2+)-induced MPT was partially decreased by calpain inhibitors. More importantly, Ca(2+)-induced inhibition of Complex I of the electron transport chain was blocked by calpain inhibitors and two Complex I proteins were identified as targets of mitochondrial calpain 10, NDUFV2, and ND6. In conclusion, calpain 10 is the first reported mitochondrially targeted calpain and is a mediator of mitochondrial dysfunction through the cleavage of Complex I subunits and activation of MPT.

Topics: 3T3 Cells; Amino Acid Sequence; Animals; Calcium; Calpain; Cyclosporine; Electron Transport; Electron Transport Complex I; Enzyme Inhibitors; Female; Humans; Male; Mice; Mitochondria; Mitochondrial Diseases; Molecular Sequence Data; NADH Dehydrogenase; Protein Sorting Signals; Protein Subunits; Rabbits; Rats; Rats, Sprague-Dawley; Submitochondrial Particles