Epilepsy affects around 50 million people worldwide, but despite being relatively common, we have yet to pinpoint a particular cell type or pathway as its main cause. One type of cell that has been the subject of a significant amount of epilepsy research is the astrocyte. Still, the question of whether astrocytes cause or protect against epilepsy remains unanswered. Fortunately, recent research that links astrocytes with the mesencephalic astrocyte-derived neurotrophic factor (MANF) may indicate a protective role.
What do we know about epilepsy?
Epilepsy affects the central nervous system (CNS), resulting in multiple changes in the brain that lead to recurrent and unprovoked seizures. Temporal lobe epilepsy (TLE) is the most common form of epilepsy and, sadly, is also highly prone to becoming resistant to the antiepileptic drugs (AEDs) currently available. The selection of AEDs available to people living with epilepsy have very different pharmacological profiles and varying degrees of efficacy when it comes to preventing seizures, as they typically only lessen the hyperexcitability rather than affecting epileptogenic processes. Current AEDs typically work by modulating ion channels or enhancing gamma-aminobutyric acid function, but this can result in adverse physiological and behavioral effects. There’s hope that a better understanding of the mechanisms underlying epilepsy will lead to better therapeutics.
Astrocytes are a double-edged sword
In looking for causes of epilepsy, many have turned their attention to astrocytes. This is because not only do we see increased numbers of astrocytes in multiple areas of the brain associated with seizure generation (e.g. at the hippocampus in TLE), we also consistently find gliotic scars, caused by reactive astrocytes, in seizure foci (the specific sites in the brain where the seizure originated).
Those reactive astrocytes are known as A1 astrocytes, which are regarded as neurotoxic, and are typically activated by neuroinflammatory microglia. A1 astrocytes can cause loss of synaptogenesis, induction of oligodendrocytes, and neuronal death. So that’s pretty damning in the case of astrocytes in epilepsy, right?
But that’s only half the story: astrocytes have another phenotype, known as A2, and in this state they’re neuroprotective, doing things like promoting neural repair and remyelination after a spinal cord injury. A2 astrocytes upregulate a number of growth factors and contribute to the resolution of inflammation and CNS repair.
So, do astrocytes have a neuroprotective effect in epilepsy and, perhaps, other diseases? As you can probably guess, the answer is not so clear-cut. Astrocytes really are a double-edged sword when it comes to normal physiological function, making untangling their role in disease even more challenging.
MANF: the protective side of astrocytes?
There’s a lot of evidence that ties astrocytes to initiating epilepsy. Astrocytes are a common feature at seizure foci and astrocytic dysfunction seems to contribute to changes in synaptic transmission and hyperexcitability. Astrocytes can also induce inflammation, which leads to epilepsy and recurrent seizures. Astrocytes are known to be the cause of high levels of glutamate (which plays a role in the initiation and spread of seizure activity) at seizure foci by down-regulating glutamine synthetase. This results in the release of glutamate to extracellular space, triggering neuronal excitation and seizures. Astrocytes might also release glutamate when they swell, a result of reduced aquaporin-4 (AQP4) expression on the perivascular astrocyte membrane.
But what about astrocytes as protective cells? For that, we need to take a look at MANF, also known as arginine-rich, mutated in early-stage tumors (ARMET). MANF is induced following endoplasmic reticulum (ER) stress and was originally identified as a secreted factor that protects rat dopaminergic neurons in vitro and prevents neurodegeneration in Parkinson’s disease.
Research shows that following epileptic and ischemic insult, MANF expression is relatively high in the cerebral cortex, hippocampus, and cerebellar Purkinje cells, while Manf mRNA expression increases temporarily following epileptic insult in the dentate granule cell layer of the hippocampus, thalamic reticular nucleus, and in several cortical areas. Research also shows that MANF exerts a host of protective effects in animal models: it promotes tissue repair, protects the heart from ischemic damage, has neurorestorative properties, and can even prevent neuron loss via inhibiting ischemia-induced apoptosis.
So if MANF were to show up in astrocytes in a model of epilepsy, it could very well have a protective effect.
Astrocytes and MANF expression
Dr. Shai Shoham, a senior member of the Alomone team, has long been intrigued by the possibility of MANF expression in astrocytes. To shed light on this fascinating topic, he used a mouse model where epileptic seizures are induced with kainate, a glutamate analog that stimulates transmission in hippocampal pathways to induce seizures. The subsequent cellular cascade results in astrocyte activation and reorganization of the hippocampal neurocircuitry – a cascade that seems to involve ER stress (which we know induces MANF release). Following the injection of kainate, Dr. Shoham used Alomone’s cutting-edge antibodies to highlight MANF and the classic astrocyte marker, glial fibrillary acidic protein (GFAP), in the hippocampal dentate gyrus (DG) and CA1.
Two hours after kainate injection, seizures developed, and MANF expression was restricted to neuronal nuclei, which closely mimicked conditions in the control mice (Figure 1). However, at four and thirty days after kainate injection, astrocytes in the hippocampal dentate gyrus molecular layer and in the stratum radiatum and oriens of CA1 began to express MANF (Figure 2).
MANF Expression in Astrocytes in a Mouse Model of Epilepsy
Figure 1. Hippocampal dentate gyrus regions in control mice and mice injected with kainate. MANF (green, vertical arrows) (#ANT-028) appears in neuronal nuclei in the granule layer (GL) and hilus (H). Several astrocytes (GFAP, red) in the molecular layer (ML, horizontal arrows) do not express MANF.
Figure 2. Hippocampal dentate gyrus regions in mice injected with kainate. MANF (green) (#ANT-028) appears in neuronal nuclei in the granule layer (GL) and hilus (H). Several astrocytes (GFAP, red) in the molecular layer (ML, horizontal arrows) express MANF.
This demonstrates that the cascade of events following kainate-induced seizures leads to an upregulation of MANF expression, specifically in astrocytes at the hippocampus. Interestingly, this astrocytic MANF expression lasts for at least 30 days and may contribute to protective mechanisms.
Are astrocytes and MANF friend or foe?
When it comes to astrocytes in general, we can say astrocytes are both friend and foe, having distinct phenotypes (A1 and A2) that are associated with an array of neurotoxic and neuroprotective effects. However, in the context of epilepsy, we can’t be certain. Astrocytes are involved in a lot of processes that seem to directly contribute to epileptogenesis. Meanwhile, they also exhibit a significant number of neuroprotective behaviors. What we do know is that following kainate-induced seizures, astrocytes start to express MANF and continue to do so for several weeks.
Dr. Shoham’s work has shed more light on these enigmatic cells and their role in epilepsy, but more research will need to be done to ascertain exactly what, if any, protective effects MANF produces.
Helping you explore astrocyte reactions to brain pathology
We’ve been producing and validating a range of antibodies in-house for decades. If you enjoyed the research discussed here, you might be interested in these primary antibodies:
Anti-MANF/ARMET Antibody (#ANT-028)
Anti-GFAP Antibody (#AFP-001) [for GFAP expression in rats]
Anti-Aquaporin 4 (249-323) Antibody (#AQP-004)
Anti-Aquaporin 4 (300-314) Antibody (#AQP-014)
Anti-Aquaporin 4 (300-314)-ATTO Fluor-594 Antibody (#AQP-014-AR)
Guinea Pig Anti-Aquaporin 4 (300-314) Antibody (#AQP-014-GP)
Anti-Connexin-43 Antibody (#ACC-201)
Anti-Connexin-37 Antibody (#ACC-204)
Anti-Calnexin Antibody (#ACS-009)
We also have blocking peptides to be used as controls for each of these antibodies. These are the original antigens we used for immunization during antibody creation:
MANF/ARMET Blocking Peptide (#BLP-NT028)
GFAP Blocking Peptide (#BLP-FP001)
Aquaporin 4 (249-323) Blocking Peptide (#BLP-QP004)
Aquaporin 4 (300-314) Blocking Peptide (#BLP-QP014)
Connexin-43 Blocking Peptide (#BLP-CC201)
Connexin-37 Blocking Peptide (#BLP-CC204)