Dr. Parag MoonSenior resident
Dept. of NeurologyGMC Kota.
1.Necrosis
Follows acute ischemia or traumatic injury.
Occurs in most severely affected areas
Abrupt biochemical collapse lead to generation of free radicals, excitotoxins.
Mitochondrial & nuclear swelling, dissolution of organelles, condensation of chromatin around nucleus.
Rupture of nuclear and cytoplasmic membranes and degradation of DNA.
Extremely difficult to treat or prevent.
2.Apoptosis Programmed cell death Seen in both acute and chronic neurologic
diseases. After acute insults occurs in areas that are
not severely affected by injury. Apoptosis occurs in penumbra, where
collateral blood flow reduces degree of hypoxia.
Chronic neurodegenerative diseases it is predominant form of cell death
Biochemical cascade activates proteases that destroy molecules that are required for cell survival.
Cytoplasm condenses, mitochondria and ribosomes aggregate, nucleus condenses & chromatin aggregates.
Cell fragments into “apoptotic bodies”
Chromosomal DNA is enzymatically cleaved to 180-bp internucleosomal fragments.
Reduction in membrane potential of mitochondria
Intracellular acidification
Generation of free radicals
Externalization of phosphatidylserineresidues
Death by Injury vs. Death by Suicide
(Necrosis vs. Apoptosis)
Caspases
Cysteine-dependent, aspartate-specific proteases.
Homologous to nematode ced-3 gene product
So far 14 members of caspase family have been identified, 11 of which present in humans.
Exist as latent precursors.
Procaspases are composed of p10 and p20 subunits and an N-terminal recruitment domain.
Active caspases are heterotetramersconsisting of two p10 and two p20 subunits derived from two procaspase molecules
Have been categorized into upstream initiators and downstream executioners
Upstream caspases are activated by cell-death signal (e.g.TNFa)
Have a long N-terminal prodomain that regulates their activation.
Upstream caspases activate downstream caspases, which directly mediate events leading to demise of cell.
Downstream caspases have short N-terminal prodomain.
Upstream caspases subclassified into two groups according to molecules modulating their activation.
Procaspases 1,2,4,5,9,11,12,13 have long N-terminal prodomain called caspase-recruiting domain (CARD).
Caspases 8 and 10 have long N-terminal prodomain called death-effector domain (DED).
Caspases 2,8,9,10-initiators of apoptosis-with a long prodomain
Caspases 1,4,5,11,12,13-cytokine activation
Caspases 3,6,7-effectors of apoptosis
Caspase 14-cytokine maturation
Upstream caspases activate in amplifying cascade executioner caspases downstream.
Executioner caspases mediate cell death by two main mechanisms: destruction and activation.
Cytochrome c-member of the mitochondrial electron-transport chain required for generation of ATP.
Important trigger of caspase cascade.
Activation occurs by release of cytochrome c is released from mitochondria into the cytoplasm.
Binds to Apaf-1 to form the apoptosome — a molecular complex consisting of cytochrome c, Apaf-1, ATP, and procaspase 9.
->Activates caspase 9, 30,52 -upstream initiator of apoptosis.
Members of Bcl-2 family are proapoptotic or antiapoptotic.
Balance between two has a crucial role in release of cytochrome c
Members of caspase family can influence balance of proapoptotic and antiapoptoticsignals.
For eg. caspase 8 and caspase 1 cleave Bid, a member of Bcl-2 family, generating a truncated fragment with proapoptotic activity.
Inhibitors interact directly with modulators of cell death.
For Eg. X-linked inhibitor of apoptosis and neuronal inhibitor of apoptosis are proteins in neurons that directly inhibit caspase 3 activity and protect neurons from ischemic injury.
P53 first arrests cell growth between G1 S
This allows for DNA repair during delay
If the damage is too extensive then p53 induces gene activation leading to apoptosis (programmed cell death)
Important in normal physiology / development– Development: Immune systems maturation,
Morphogenesis, Neural development
– Adult: Immune privilege, DNA Damage and wound repair.
Excess apoptosis– Neurodegenerative diseases
Deficient apoptosis– Cancer
– Autoimmunity
Difference between acute and chronic neurologic diseases is magnitude of stimulus causing cell death.
Greater stimulus in acute diseases results in both necrotic and apoptotic cell death
Milder insults in chronic diseases initiate apoptotic cell death.
Activation of caspases1, 3, 8, 9, and 11 and release of cytochrome c seen in cerebral ischemia,
Mice that express a dominant-negative caspase 1 construct or that are deficient in caspase 1 or caspase11 have significant protection from ischemic injury.
Mice T/T with broad caspase inhibitor/semiselectiveinhibitors of caspase 1 & 3 protect from ischemia.
Pattern of combined necrotic and apoptotic cell death after ischemic or traumatic injury.
Necrotic cell death-core of infarction.
Ischemic penumbra-activate caspase cascade.
TBI-apoptosis-related changes in neurons like presence of DNA strand breaks, caspaseactivation, increased Bax, p53 expression.
Intraventricular administration of caspase-3 inhibitor z-DEVD-fmk before injury reduces cell death and improves symptoms.
Mice expressing dominant-negative inhibitor of caspase-1 show reduced brain damage and free radical production after TBI.
Intraventricular infusion of NGF in rats resulted in improved learning and memory and decreased death of neurons in TBI.
Cyclosporin A protects against synaptic dysfunction and cell death in rodent models of TBI.
In rodents, SCI can be prevented by glutamate-receptor antagonists.
Degenerating neurons show aggregates of hyperphosphorylated tau protein & excessive calcium-mediated proteolysis and oxidative stress.
Increased DNA damage & caspase activity
Alterations in expression of apoptosis-related genes such as Bcl-2 family members, Par- 4 and DNA damage response genes.
Marked decrease in expression of anti-apoptotic gene called NCKAP1
Amyloid-β sensitizes neurons to death involves membrane lipid peroxidation
Impairs function of membrane ionmotiveATPases and glucose and glutamate transporters resulting in membrane depolarization, ATP depletion, excessive calcium influx and mitochondrial dysfunction.
APP-substrate for caspase-3 Caspase-mediated cleavage of APP release a
carboxy-terminal peptide called C31, a potent inducer of apoptosis
Presenilin-1 mutation leads to disturbances in calcium homeostasis in endoplasmic reticulum (ER) such that more calcium is released in neurons.
Neurotrophic factors,cytokines known to prevent neuronal apoptosis
Increased oxidative stress and mitochondrial dysfunction in dopamine neurons are central to disease.
Deficit in Mitochondrial Complex I which may arise from or contribute to increased cellular oxidative stress.
Environmental and genetic factors sensitizes neurons to oxidative stress.
Monkeys and people exposed to toxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) show Parkinson’s-like symptoms
Apoptosis-related DNA damage and gene activation seen in death of dopamine neurons
Levels of Par-4 are selectively increased
Suppression of Par-4 expression protects dopamine neurons against death
Caspase-1 inhibition, glial cell-derived neurotrophic factor (GDNF) can protect dopamine neurons.
Expression of mutant α-synuclein in cultured cells promotes apoptosis.
Impaired mitochondrial function and excitotoxicdeath may be central to disease.
Studies of Lymphoblasts from patients with Huntington’s disease showed increased stress induced apoptosis associated with mitochondrial dysfunction
Increased caspase-3 activation.
Mutant huntingtin in cultured cells induces caspase8-dependent apoptosis.
Huntingtin can be cleaved by caspases, which may promote protein aggregation and neurotoxicity.
Inhibition of caspase-1 was reported to slow disease progression in mouse models.
Increased oxidative stress, overactivation of glutamate receptors and cellular calcium overload.
Autoantibodies against voltage-dependent calcium channels.
Mutations in antioxidant enzyme Cu/Zn-superoxide dismutase (SOD)
Increased peroxidase activity.
In ALS DNA starts to fragment between nucleosomes(a sign of nuclear apoptosis) in neurons within the spinal-cord anterior horn and motor cortex.
Levels of Bax, but not Bcl-2, are increased in spinal-cord motor neurons of ALS patients.
Vitamin E, melatonin, resveratrol, carnosine, coenzyme Q10
Vit E-slightly delays disease progression in patients with moderately severe AD and PD and can improve the cognitive function in AD patients
Some clinical trials showed vitamin E actually aggravated AD
Polyphenol with powerful antioxidant properties
Abundant component of red wine
May cause increased neuroprotective activity through activation of sirtuin 1 (SIRT1)
SIRT1 exerts antiapoptotic and neuroprotectiveeffects by deacetylating transcription factors, such as the tumour suppressor p53, the FOXO family of proteins (also called FKHR, a member of the Forkhead family of transcription factors), and NF-B.
SIRT1 activation also has anti-aging properties in invertebrates.
Radical scavenger, anti-apoptotic and anti-aging properties.
Prevents mitochondrial calcium overload, mitochondrial depolarization, ROS formation, opening of mitochondrial permeability transition pore (PTP) that precedes Cyt c release.
Clinical trial in PD patients, melatonin (3mg) did not improve motor dysfunction abnormalities but some beneficial effects noted in quality of sleep.
In AD patients-beneficial effects of melatonin (3 to 9 mg) on memory loss are unclear.
Clinical trials showed that combining carnosine (daily dose of 1.5 g for 30 days) with the conventional treatment for PD significantly improves neurological status and locomotor performance.
Could be used as an anti-aging treatment as well as in AD treatment.
No clinical trials of using this drug in AD
No publication of data on the effects of carnosine on cognitive function.
Cofactor of electron transport chain.
Prevents mitochondrial dysfunction
Antioxidant properties.
Evidence that it decreases with aging in both human and rat tissues.
Cognitive performance of patients with AD improved when treated with an antioxidant together with acetylcholinesterase inhibitors.
Phase II clinical trials reports potentially beneficial effects in CoQ10 in PD.
Ongoing research in HD, ALS, PSP.
Attenuate neurotoxicity in neuronal cell preparations.
Temporarily rescue neurons since these surviving neurons are in a dysfunctional state due to release of pro-apoptotic proteins.
Two types of calpains u and m-calpain
Proteases when activated induce degradation of cellular substrates such as cytoskeletal proteins, membrane proteins, phosphatases.
Increased calpain immunoreactivity seen in senile plaques of AD and PD brains.
P35->p25->activation of CDK5-> phosphorylation of myocyte enhancing factor2(MEF-2)->apotosis.
CDK5 inhibitor roscovitine- antiapoptotic and neuroprotective effects in several models of neurodegeneration
Rodent and cell culture models calpaininhibitors such as calpeptin, MDL-28170 and PD150606 shown to prevent neuronal death and restore cognitive function in AD models.
Ability to phosphorylate a variety of substrates.
Lithium -direct & reversible GSK-3 inhibitor
Also inhibits calpain/CDK5 pathway & modulates NMDA receptor.
Also favours autophagy.
Contradictory results in clinical trials in AD and ALS.
Newer compounds-Paullones, indirubines, thiazoles, aminopyrimidines and bisindol-maleimides.
ATP inhibitors.
Developed by Glaxosmithkline and AstraZeneca.
G1/S blockers-flavopiridol, kempaullone and roscovitine showed neuroprotectiveproperties in neuronal cell cultures.
Anticancer drugs.
More side effects.
Anti-inflammatory properties, decreases microglia activation, free radical formation, prevent excessive intracellular calcium entry via NMDA receptors.
Varied penetration via blood brain barrier (atorvastatin, lovastatin)
Tried in PD, DLB, ALS, MS with varied results.
Mitigate mitochondrial calcium overload, prevent ROS production, inhibit cyt c release, increase in neurotrophin production
Daily ibuprofen (50 mg/kg) in APP23 AD mice models and in humans caused increase in cognitive activity
R-flurbiprofen –decreased learning impairments in AD
Discouraging results in clinical trials.
Rosiglitazone, troglitazone-neuroprotectiveeffect against -amyloid-induced cell death.
Capacity to stop inflammatory gene expression in peripheral immune cells
Reduced microglial and astrocyte activation.
Tried in AD, MS
Prevents release of proapoptoticmitochondrial proteins such as cytochrome c into the cytosol
Upregulates Bcl-2 expression
Reduces cleavage of Bid, a protein of the Bcl-2 family with pro-apoptotic properties
Anti-inflammatory properties through modulation of immune cytokines
Tried in AD,PD,HD,ALS
Calcium overload which leads to apoptotic cell death
Memantine-uncompetitive NMDA receptor antagonist that blocks with low affinity
Does not interfere with physiological activity of glutamate in learning and memory processes
Dizocilpine, selfotel, aptiganel, remacemide,licostinel-unacceptable side effects.
NGF role in maintenance of cholinergic neurotransmitter systems in cholinergic forebrain neurons
CERE-110 is a genetically engineered replication defective adeno-associated virus serotype 2 (AAV2) vector
Contains full-length human -nerve growth factor cDNA.
Phase II study in AD BDNF may enhance differentiation and survival of
dopaminergic neurons in substantia nigra. Several small molecules targeted to BDNF
receptors are being developed.
thanks
Apoptosis and Caspases in Neurodegenerative Diseases: Robert M. Friedlander: N Engl J Med 348;14:april 3, 2003
Antiapoptotic drugs:A Therapeutic Strategy for the Prevention of Neurodegenerative Diseases;CurrentPharmaceutical Design, 2011, Vol. 17, No. 3 233
Apoptosis In Neurodegenerative Disorders: Mark P.Mattson; Nature Reviews | Molecular Cell Biology Volume 1 | November 2000 | 121
Neuroprotection in Progressive Brain Disorders; Ruth Djaldetti, Nirit Lev, Eldad Melamed; IMAJ . Vol 5 . August 2003
Gene Transfer of Baculoviral p35 by Adenoviral Vector Protects Human Cerebral Neurons from Apoptosis;DNA AND CELL BIOLOGY;Volume 23, Number 8, 2004
Apoptotic and antiapoptotic mechanisms in stroke; Cell Tissue Res (2000) 301:173–187