Block 3: CNS disorders and treatments Flashcards
Describe the neuropathology of Alzheimer’s Disease.
The two main pathological hallmarks of AD are amyloid-beta plaques and neurofibrillary tangles.
Extracellular amyloid plaques contain an insoluble 42-residue peptide (beta amyloid 42) in their core, surrounded by neurites, microglia, and astrocytes (including structural abnormalities such as enlarged mitochondria, liposomes, and impaired filaments). These plaques may take years to fully form. They are seen across many neurodegenerative diseases, and are also seen in non-diseased, ageing brains (known as senile plaques), however, they are much more prevalent in AD. These plaques largely aggregate around the temporal lobes and limbic system structures, as well as the prefrontal cortex. This can be shown in PET scans using Pittsburgh compound B (radiolabelled thioflavin derivative) which associates with the AB plaques.
In the healthy brain, 90% of AB-peptide is in the form of AB40, and is derived by the cleavage of a much larger protein (amyloid precursor protein APP). It is normally cleaved by a-secretase to give this AB40 form (soluble and can be easily removed), however, abnormal cleavage can be caused by B- or y-secretase, leading to the formation neurotoxic peptide AB42 (insoluble). AB42 can aggregate into oligomers. APP mutations can lead to favourable cleavage by beta-secretase, resulting in increased oligomerisation; PSEN1/PSEN2 mutations can lead to favourable cleavage by gamma-secretase (same outcome). Amyloid-B plaque formation can impair synaptic function between neurons, boosted LTD and suppressing LTP. Additionally, the aggregation of oligomers into insoluble beta-sheet amyloid fibrils can trigger local inflammatory responses which, over time, causes oxidative stress and biochemical changes which ultimately lead to neuronal cell death and the deposition of plaques and tangles.
Neurofibrillary tangles are dense bundles of fibres in the cytoplasm of neurons, containing a highly polymerised form of a cytoskeletal protein Tau. These occur in many chronic brain diseases (not just in AD), but are more prevalent (same as AB plaques). The hippocampus and parieto-temporal regions of the cerebral cortex are particularly susceptible (helps to explain early symptoms). Tau proteins stabilise the structure of microtubules – they become hyperphosphorylated in AD, causing them to dissociate from the microtubule and become tangled (initially form paired-helical filaments which associate together). This results in a de-stabilisation of the structure of the microtubule, preventing proper axonal transport from taking place, and leading to neuronal cell death. Using the same PET technique as previously discussed for observing AB plaques, neurofibrillary tangles can be imaged in the diseased brain – this reveals that some region of the brain have tangles but no plaques, suggesting that the plaques do not cause the tangles as proposed by the amyloid cascade hypothesis.
Outline genetic predisposition to Alzheimer’s.
There are two forms of AD- familial and sporadic. Familial AD is the less common form, and is believed to be mostly genetic, whereas sporadic (later onset) AD has complex genetic epidemiology mixed with other risk factors. Familial AD is attributed to presenilin 1 (chromosome 14) and presenilin 2 (chromosome 1) mutations (PSEN1/2).
Sporadic AD is more associated with APP gene (chromosome 21) mutations, as well as APOE4 variation of gene APOE (chromosome 19). Apolipoprotein E is involved in neuronal repair and growth (as well as cholesterol transport). Common amino acid variations also shown to be associated with predisposition to Alzheimer’s disease (also associated with senile AB plaques and neurofibrillary tangles). In vitro, apolipoprotein E has a high affinity to AB42 peptide, increases its formation, and interferes with its removal. There are three major APOE alleles- APOE2, 3, and 4 – the majority of the population has the E3 variant (~78%), and the minority with E2 (6%), E4 (16%). APOE2 has been shown to give a certain degree of protection from AD. These alleles differ by just two amino acids. Normal cholesterol transport is required for the removal of B-amyloid protein from the CNS, so APOE4 causes accumulations of B-amyloid and binding of B-amyloid to the tau protein of neurofibrillary tangles. Heterozygous for APOE4 = 2 times risk of developing sporadic AD; homozygous = 5 times risk.
Outline the treatment options available for AD.
Treatments almost all target the symptoms, rather than the disease (also, before the disease becomes symptomatic it causes a lot of damage). Some treatments target the cholinergic and glutamate systems in an attempt to help memory issues. Tacrine, donepezil, and Aricept block Ach breakdown by inhibiting acetylcholine esterase. Memantine is an NMDA receptor antagonist, shown to reduce some clinical symptoms with moderate to severe AD (staves off excitotoxicity). The problem with these is that they have huge side effects.
More contemporary approaches aim to target the APP secretase enzymes which produce AB42, or to reduce the activity of AB42 itself. B-site APP cleaving enzyme (BACE) inhibitors should in theory be able to block beta-secretase, and therefore prevent the formation of excess AB42. This can also be applied to gamma-secretase, but these are not as good targets as they produce toxic side effects (gamma-secretase is important for lymphocyte development and intestinal structures). BACE inhibitors are not associated with these side effects, but it is difficult to get them across the BBB.
Glycosaminoglycans (GAGS) bind AB in solution which leads to plaque formation. GAG mimetics compete with GAGS to block this aggregation process. Ovine colostrinin is one such example which has been shown to improve learning in AD in animal models. SALAs (selective amyloid lowering agents) are a new class of anti AB drugs which target mild AD. Tarenflurbil was a very promising agent, but was one of the largest drug trials to ever fail (worked in animal models). This could be because blocking AB is not an appropriate drug target, or because tarenflurbil itself is a weak pharmacological agent (there is debate on this).
Outline the 5 stages of progression of Parkinson’s Disease.
1) Unilateral tremor, or difficulty performing simple manual functions, leaning to affected side (tremor often improves or disappears with purposeful function).
2) Bilateral involvement with early postural changes, slow shuffling gait with decreased excursion of legs.
3) Pronounced gait disturbances and moderate generalised disability, postural instability and tendency to fall.
4) Significant disability, limited ambulation with assistance.
5) Complete invalidism, patient confined to bed or chair, cannot stand or walk even with assistance.
Describe the pathology of PD.
Lewy bodies are spherical neuronal inclusions (easily identified post-mortem), and are a defining feature of PD. These Lewy bodies can be found in the cytoplasm of surviving neurons, and are comprised of many proteins including a-synuclein. Under a microscope they are easily identified as pink spheres with pale halo around them. a-synuclein is a synaptic protein present in presynaptic terminals, possibly involved in neurotransmitter storage and release, vesicle recycling, and synaptic plasticity. Mutations in the synuclein gene has been associated with familial PD. Lewy bodies have been found throughout the brain in PD post-mortems. It is proposed that they are first deposited in the olfactory bulb and lower brainstem, then in the substantia nigra before progressing to the cortex (would explain the loss of smell and REM sleep disorders in onset before motor impairments).
Depletion of neurons in the SNpc in very pronounced in people with PD. Up to 60% of nigral neurons are lost before any motor impairments appear. This delay is accounted for by increased dopamine production by surviving neurons (compensatory) or by upregulation of dopamine receptors in target striatal neurons (current avenue of research). Nigrostriatal tract (one of 4 dopaminergic pathways in the brain) degenerates leading to less than 20% dopamine levels in basal ganglia. The nigrostriatal pathway is the one associated with motor, and is the one associated with causing the pathology of PD (originates in the SN and projects to the striatum). The mesolimbic pathway is associated with emotional behaviours (cell bodies from the ventral tegmental area project to the nucleus accumbens and amygdala). The mesocortical pathway is also associated with emotional behaviours (cell bodies within ventral tegmental area project to the frontal cortex). The tuberohypophyseal pathway project from the ventral hypothalamus to the pituitary (regulate pituitary secretions).
Other pathologies associated with PD are mitochondrial dysfunction (defect of mitochondrial complex 1 confined to the substantia nigra – linked with oxidative stress and elevated brain iron levels); and microgliosis (inflammation and change of cytokine levels in substantia nigra and CSF).
Discuss the various treatments available for PD.
Levodopa is one of the most common first-line treatments for PD. Dopamine is produced endogenously in the brain from tyrosine, which is converted to DOPA, then dopamine. Levodopa is a synthetic form of dopamine precursor DOPA, and can be converted to dopamine in dopaminergic neurons by DOPA decarboxylase (prescribed instead of dopamine, because dopamine cannot cross the BBB). It is given with DOPA decarboxylase inhibitors (e.g. carbidopa), which cannot cross the BBB, to prevent peripheral side-effects. As PD progresses, levodopa effectiveness decreases, and more continuous dopaminergic stimulation is required to maintain consistent physiological activity.
Dopamine agonists are effective in controlling PD symptoms, however there is a wide range of side-effects. They don’t show fluctuations in efficacy which are seen with levodopa but they may cause somnolence, hallucinations, and predisposition to compulsive behaviours.
MOA-B inhibitors inhibit the breakdown of extraneuronal dopamine in the brain. Therefore lack the unwanted side effects of non-selective MAO inhibitors (used in treatment of depression). Clinical trials show that a combination of MAO-B inhibitor Selegiline and levodopa are more effective than levodopa alone
Other treatments include:
- Neural transplantation (injection of foetal neuroblasts into the striatum). Up to 5 foetuses may be required for sufficient neuroblasts. Some transplants have been relatively successful, and dopamine neurons have become functional again (but side effect of serious dyskinesia).
- Gene therapy aims at increasing synthesis of neurotransmitters and neurotrophic factors (for example, expressing tyrosine hydroxylase or dopa decarboxylase; or increasing synthesis of GABA in subthalamic nucleus by overexpressing glutamic acid decarboxylase, to reduce excitatory input).
- Deep brain stimulation (DBS) involves implanting an electrode into the brain (subthalamic nucleus or globus pallidus). This is used to alleviate motor symptoms in patients with severe motor complications.
Describe how stroke damages neural tissue.
Lots of different pathways contribute to brain injury, these are not independent of each other (e.g. cytotoxic oedema, excitotoxicity, oxidative stress), but feed into one another in a positive feedback loop to amplify the insult. The major cause of cell death is energy failure (without oxygen and glucose, cells cannot produce ATP, and therefore cannot sustain ion pumps). This results in inappropriate ion gradients, and significant accumulations of sodium and calcium, resulting in swelling, degeneration of organelles, loss of membrane integrity, and dissolution of the cell. Increase in synaptic glutamate results in overstimulation of NMDA, resulting in downstream cascading effects due to intracellular calcium (excitotoxicity). Damage from oxidative stress to the BBB can result in inflammation (causing further oxidative stress). The contribution of these pathways to brain damage following a stroke varies throughout time.
The result of reintroduction of oxygen when blood flow is restored is the generation of lots of reactive oxygen species (due to reduction in endogenous antioxidant protection). Because the brain is very lipid rich, it is sensitive to oxidative damage. It also has high levels of iron which act as pro-oxidant during stress and high oxygen consumption at basal levels. The main sources of ROS in the brain are mitochondrial respiration chain; NADPH oxidases, and xanthine oxidases (as well as nitrogen oxidases). ROS can then cause DNA fragmentation, apoptosis, protein denaturing, lipid peroxidation, and inflammation.
Hypoxia, ROS, and shear stress result in blood clotting, platelet aggregation, and cytokine release in the blood vessel lumen. P-selectin is translocated onto the surface of platelets, and platelets and leukocytes aggregate. Compliment is activated, releasing AA metabolites. Upregulation of E- and P-selectin provides a site for leukocyte binding on the vascular wall. A loss in nitric oxide occurs due to ROS generation (disruption of vascular tone), which enhances leukocyte and platelet aggregation. MMP activates, leading to BBB breakdown. Histamine is released, which can further contribute to BBB leakiness. Damaged cells release ATP which acts as an early pro-inflammatory signal, leading to the production of cytokines and chemokines (activates microglia, which leads to production of more inflammatory mediators and ROS). This all creates a snowball effect.
Outline the current treatment options for stroke.
Treatment within a dedicated stroke unit (multidisciplinary team of experts) has been shown to reduce mortality by 3%, dependency by 5%, and need for institutional care by 2%. Antiplatelet therapies (e.g. aspirin) have been shown to reduce death and dependency, as well as recurrence of stroke. The best pharmacological treatment is recombinant tissue plasminogen activator (rt-PA, a thrombolytic). rt-PA is the only currently approved drug for acute ischaemic stroke – it activates plasmin to degrade fibrin clots (clot-busting drug). Although it doesn’t hugely increase chances of survival from stroke, it has been shown to hugely decrease dependency in stroke survivors. Thrombolytics must be administered within 4.5hrs of symptom onset. People often don’t arrive at hospital within this window, as it is not as obvious as a heart attack for instance. Cost per treatment of thrombolytics is £300-600. Chances of excellent outcome of treatment is increased by 2.3 times if treated within 90 minutes (odds fall linearly with time from onset). They must confirm that the patient is suffering from an ischaemic stroke to treat them with thrombolytics, if it were a haemorrhagic stroke it would be a terrible treatment. New devices which physically remove clots from the blood stream have been recently developed (thrombectomy). These devices have been shown to improve chances of good outcome after recovering from stroke when treated fast, but also when patients don’t get treated as fast. Novel drugs which aim to assist thrombectomy are currently being developed.
What are miRNAs and extracellular vesicles?
miRNAs (micro-RNAs) are considered the fine-tuners of gene expression (increase in their expression leads to inhibition of translation of their target genes). These are short, non-coding RNAs (typically 20-22 nucleotides) which are held within extracellular vesicles. The seed sequence (conserved heptametrical sequence, mostly situated at positions 2-7 from the miRNA 5’-end) is very short, meaning that one miRNA can target multiple genes – these genes may contribute to similar pathways and processes or can be completely distinct. This means that by delivering one miRNA you can achieve polytherapy outcome from a single intervention.
Exosomes are small extracellular vesicles (<150nm diameter), and are derived from multivesicular endosomes (within cells). Microvesicles are larger and result from budding of the membrane (have markers of the origin of the cell from which they are derived; typically >500nm diameter). These systems are important for many physiological functions within the body, including inflammation and have been implicated in a range of specific diseases including cancers, cardiometabolic diseases, neurological diseases, and infectious diseases. They can be used as endogenous therapeutics in the response to disease, or as biomarkers for identification of specific diseases.
Describe how miRNAs can be used as a biomarker for stroke risk and as a treatment for stroke.
Clinically, altered miRNA expression has been observed in circulating plasma serum and CSF of stroke patients. Preclinical models have found that miRNA levels change within the brain tissue itself after stroke. Several studies have profiled miRNA levels at pre-stroke (risk factors), acute stage stroke, and chronic stage stroke. Many of the miRNA levels in pre-stroke profiles are associated with diabetes, hypertension, and atherosclerosis. A whole host of miRNAs linked to excitotoxicity, oxidative stress, inflammation, BBB damage, and apoptosis are seen in acute stroke profiles. miRNAs linked with neurogenesis and angiogenesis are observed in patients with chronic stage stroke. These have been found by cross-referencing the DNA targets of these miRNAs with databases to provide predictive and validated targets. These gene targets can be sorted by the processes which they will be involved in.
A systematic review aiming to determine the usefulness of miRNAs as biomarkers for acute ischaemic stroke was conducted in 2018 by Dewdney et al. fully analysed 8 studies which included almost 600 cases and over 400 healthy controls, and found 22 miRNAs were differentially expressed (12 upregulated, 10 downregulated). Only one of these miRNAs was differentially expressed in at least 2 studies, showing that there is considerable heterogeneity across the studies. It is clear from these studies that more research is required to evaluate the diagnostic potential of miRNAs. However, these looked at circulating miRNA expression rather than those exclusively in extracellular vesicles. Looking solely at those which are expressed in EVs may overcome some of the disparity seen in these studies.
A preclinical study, by Chen & Chopp (2018), investigated how these miRNAs could be used to deliver therapeutic agents to the brain for stroke recovery. This study used rat models (n=6) which were subjected to experimental stroke (transient middle cerebral artery occlusion tMCAO), and intravenously injected extracellular vesicles derived from mesenchymal stem cells (MSCs) 24 hours later (it was hypothesised that MSCs, a common treatment for stroke recovery, elicit their effects via EVs). It was found that these vesicles freely passed through the BBB and interacted with target cells to transfer cargo via endocytosis, direct fusion, or binding through receptor-ligand interaction. These can then induce a host of effects which can be used to initiate neuro-regenerative processes, including neurogenesis (as well as angiogenesis, oligodendrogenesis, and synaptogenesis), axonal remodelling, vascular remodelling, and reductions in inflammation; all of which promote neuroprotection and neuro-restoration to improve functional outcome. The results determined that many MSC benefits are indeed mediated by miRNAs found within EVs
Another more recent preclinical study by Li et al. (2021) looked at cerebral endothelial cell (CEC)-derived EVs rather than MSC-derived. This study used a more clinically relevant stroke model – an embolic middle cerebral artery occlusion model (eMCAO; blood clot is injected, more representative of an ischaemic occlusion). This therapy was paired with thrombectomy, as well as tested independently, to test if it assists. It was found that when paired with thrombectomy, the CEC-derived EV treatment significantly improved outcome. They also found that this agent would not increase the chance of haemorrhage (thrombectomy alone increases the chance of haemorrhage). The combined therapy found improvements in neurological function and reduction in infarct volume of ~37% (compared to control).
What are partial and generalised epileptic seizures?
Partial (focal) seizures arise in a specific region in the brain (within the temporal lobe). Simple partial seizures do not impair awareness; complex partial seizures do; and partial seizures which affect the contralateral side are known as secondary generalised seizures.
Generalised seizures arise spontaneously and simultaneously in both hemispheres. these seizures take more diverse forms, such as absence seizures, myoclonic seizures, clonic seizures, tonic seizures, tonic-clonic seizures, and atonic seizures.
There are also unclassified epileptic seizures which don’t fit into the two categories.
What is known about the brain mechanisms underlying epilepsy?
Epilepsy probably involves ion channel dysfunction and/or neurotransmitter imbalance. Action potentials are largely governed by influx of sodium and efflux of potassium via VGICs. In EEGs of people with epilepsy, we often observe abnormal spikes which are known as paroxysmal depolarising shifts (PDSs) – this is an abnormal AP, which involves the additional input of calcium channels and LGICs that are sensitive to glutamate and GABA. This results in prolonged depolarisation of the membrane, with many spikes in depolarisation. After which, there is a longer lasting refractory period. However, it is unknown what causes this epileptic discharge, or what turns one isolated epileptic discharge into an epileptic seizure.
Whilst ion channels may account for the abnormal discharges in individual neurons, excitability of networks are governed by neurotransmitters. Glutamate and GABA are the neurotransmitters which are most characterised in epilepsy. One potential cause of epilepsy could be a simple neurotransmitter imbalance between glutamate and GABA, resulting in imbalance of excitatory and inhibitory signals (disruption to homeostasis). It can therefore be hypothesised that either under-release of GABA, or excessive release of glutamate could cause epileptic seizures.
Compare idiopathic and symptomatic epilepsy and the proposed mechanisms underlying each.
Epilepsy can also be classified aetiologically as either idiopathic or symptomatic. Idiopathic epilepsies are mainly generalised seizure disorders with presumably genetic origins – most are believed to have complex polygenic inheritance which makes identification of causative genetic mutations difficult. Some of these have mendelian inheritance (single gene). An example of a monogenic epilepsy is severe myoclonic epilepsy of infancy (SMEI; AKA Dravet syndrome) – it presents at ~6 months of age with generalised clonic seizures (absence and focal seizures may develop later). Seizures typically unresponsive to antiepileptic drugs; children develop cognitive, behavioural, and motor impairments. In 70-80% of cases of SMEI, there is a mutation in SCN1A (encodes the alpha subunit of a VGNa+ channel, resulting in a loss of channel function – would expect it to increase function, but that’s not the case). The current belief as to why this LOF causes seizures is because it is predominantly expressed in inhibitory interneurons (loss of inhibitory control) – this would also explain why it is unresponsive to drugs which block ion channels (make it worse by blocking residual channel function).
Symptomatic epilepsies arise from a known abnormality (i.e. can often be seen in an MRI or CT scan). These are often called acquired epilepsies, and are often focal seizures which arise from an area of injury. Feed-forward and feedback inhibition via inhibitory interneurons are crucial for preventing excessive excitation in many neural circuits. Feed-forward inhibition (excitatory neuron synapses to inhibitory interneuron which feeds back to the same cell the excitatory neuron synapses with); feedback inhibition (output from excited cell also excites inhibitory interneuron which feeds back to the same cell). It is likely that these inhibitory interneurons are impaired by the lesions. There are several theories as to how this occurs:
1) Upon brain injury, these inhibitory interneurons are selectively lost.
2) The excitatory interneurons that synapse to these inhibitory interneurons are lost, resulting in loss of inhibitory drive.
3) “sprouting” of axons forms abnormal connections with excitatory neurons in the network.
4) Neurogenesis (in specific areas of the brain, such as the hippocampus) – new excitatory neurons are specialising from precursor cells.
Outline the current treatment options for epilepsy.
Phenytoin and carbamazepine are classic sodium channel blockers which show preferential binding to inactivated state of the channel, exerting a use- and frequency-dependent block. Block is enabled by use (have to use the channel to bring it to its inactivated state in order for the drug to bind); frequency of use enhances the block (more frequent activation will result in more channels being in the inactivated conformation, and therefore the efficacy of the drugs increases with frequency of activation). This means that the drugs selectively inhibit high frequency APs (e.g. during a seizure). VGNaC blockers are the most numerous family of anti-epileptic drugs.
Phenobarbital (PB) and diazepam (DZP) are drugs which bind to distinct sites on the GABAA receptor complex to enhance its response to the binding of GABA (positive allosteric modulators). They can also activate the receptor in the absence of GABA. Barbiturates such as phenobarbital prolong the during of the channel opening, whereas benzodiazepines such as diazepam increase the frequency that they open.
Gabapentin and pregabalin are P/Q-type calcium channel blockers. These are involved in neurotransmitter release at the synapse, so blocking them can prevent excitatory neurotransmitters being released. They are sometimes used for epilepsy, but more commonly in the treatment of neuropathic pain.
Perampanel is a selective, non-competitive antagonist at AMPA glutamate receptors. It binds at the boundary of the extracellular and transmembrane domains to affect conformation of the receptor. It is also very potent – it is well tolerated unlike most glutamate receptor antagonists which block fundamental physiological brain functions leading to pronounced adverse effects.
Levetiracetam and brivaracetam bind selectively to synaptic vesicle glycoprotein 2A (SV2A) to inhibit it. SV2A is involved in neurotransmitter release at the synapse via calcium entry, and is found in pre-synaptic vesicles.
Valproate (AKA valproic acid or sodium valproate) is one of the oldest antiepileptic drugs, and still no one knows how it works. Proposed ideas include sodium channel blocking, T-type calcium channel blocking, positive modulation of GABAA receptors, promotion of GABA synthesis and inhibition of GABA metabolism, and reduced brain aspartate concentration. It is also licensed for the treatments of bipolar disorder and migraine (and under investigation in schizophrenia, HIV infection, and cancer). It may inhibit a whole range of proteins expressed in the brain epigenetically.
What are the effects of general anaesthetics and what brain areas are involved?
General anaesthetics aim to provide balanced anaesthesia (amnesia, analgesia, and relaxation). They are used in combination with other drugs such as neuromuscular blockers, sedatives, and anxiolytics. Amnesia is driven at the hippocampus and amygdala; anaelgesia by suppression of nociceptive inputs at the spinal level (substantia gelatinosa); unconciousness is driven by the reticular activating system in the brainstem and thalamocortical tracts; and muscle reflex is suppressed at the level of spinal interneurons.
