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陳凡、賀星惠、高青、欒國明、李天富

Introduction Epilepsy is a common condition effecting people of all ages, gender, and socioeconomic status. It is the third most common neurological cause of years lived with disability in the world . Epilepsy is a prototype neuro- psychiatric illness between the interface of neurology and psychiatry. Therefore, treatment of comorbidity associated with epilepsy could contribute to the thera-peutic effect of epilepsy and improve the quality of life. More than one third of patients with epilepsy are pharmacoresistant , and apart from those who are candi-dates for resective surgery, most patients will continue to suffer recurrent seizures and comorbidities compris-ing of neurologic, cognitive and psychiatric symptoms . Therefore, novel solid biomarkers for the prediction, diagnosis, and treatment of epilepsy and comorbidities associated with epilepsy are crucial to improve the quality of life in patients with epilepsy.

Adenosine, as an endogenous neuromodulator, plays a crucial role in inhibition of seizures . ADK is the chief enzyme in adenosine metabolism under baseline condi- tions and crucial to maintain adenosine homeostasis. ADK activity keeps adenosine levels low. Slight chang-es in enzyme activity result in major changes of adenos-ine concentration. Adenosine is an inhibitory neuro-modulator released during epileptic seizures, and demonstrated an important role to stop seizures, postic-tal refractoriness, and inhibition of the development of epileptogenesis . Dysfunction of adenosine system has been demonstrated as one of the mechanisms underly-

ing for comorbidities associated with epilepsy. In addition, focal augmentation of adenosine in the brain not only inhibits recurrent seizures but also improve comorbid symptoms associated with epilepsy. Overex-pression of ADK has recently proved to lead to cogni-tive and psychiatric symptoms associated with epilepsy . Adenosine kinase has been extensively studied in experimental epilepsy models and in patients with pharmacoresisitant epilepsy such as mesial tempo- ral lobe epilepsy (MTLE) , tumor related epilepsy ,Rasmussen encephalitis (RE) , focal cortical dysplasia (FCD) and Sturge-Weber syndrome (SWS) . Overex-pression of ADK plays a significant role in the epileptic seizures and epilepsy development . Therefore, ADK is regarded as a specific diagnostic and treatment target for epilepsy by activation of multiple adenosine recep-tors-dependent pathways .

Developmental studies performed in mice indicate that there is a switch from neuronal expression during the perinatal period to almost total astrocytic expression in the adult brain. Interestingly, strong neuronal expres-sion of ADK has been detected in human fetal brains (gestational week 13; temporal cortex). Dynamic changes in ADK gene transcription during early postna-tal brain development was also documented, and binding of transcription factor specificity protein 1 to the ADK promoter influences the regulation of ADK expression .

The overlap of maladaptive changes in adenosine homeostasis suggests common pathogenic mechanismsfor epilepsy and a comorbidities associated with epilep-sy. Overexpression of ADK and adenosine deficiency is a common pathological hallmark in patients with pharmacoresistant epilepsy caused by a wide range of neurodegenerative conditions . Therefore, therapeutic adenosine augmentation might have the potential to treat recurrent epileptic seizures and comorbidities associated with epilepsy in these neurodegenerative diseases such as MTLE, RE, FCD and SWS.

1. Overexpression of ADK in RE

RE is a rare progressive inflammatory disorder of uncertain etiology that characteristically occurs in children who present with refractory seizures, cognitive deterioration, and progressive hemiparesis, resulting from dysfunction of one cerebral hemisphere. RE is associated with hemispheric atrophy, focal epilepsy (epilepsia partialis continua, EPC), cognitive deteriora-tion, and progressive neurologic deficits that result from progressive loss of function subserved by the involved cerebral hemisphere. Radiologically, charac-teristic magnetic resonance imaging features are areas of cortical hyperintense T2/fluid-attenuated inversion recovery signal and progressive atrophy in the affected cerebral hemisphere with mild or severe enlargement ventricle (Fig. 1A-C, circles). The pathologic features of RE are well described as lymphocytic infiltrates (perivascular lymphocytic cuffing), microglial nodules, neuronal destruction, and gliosis of the affected hemi-sphere.

Over expression of ADK has been indicated to play a crucial role in the recurrent seizures and the develop-ment of RE . In control (autopsy or surgical control cortex) white matter, immunostaining indicated that weak ADK expression only in sparse glial cells and lack of ADK immunoreactivity in neuronal cells . In addition, our recent work showed that there is no obvi-ous difference in ADK expression between children (more than 3 years) and adult autopsy control cortex.Therefore, further study is needed on younger children for the developmental and age-related differences. In RE specimens, there were reactive astrocytes in cortical gray matter (Fig. 1D, arrows) and white matter (Fig. 1E, arrows) within the lesion area, which are characterized by a hypertrophic morphology with larger soma and

increased length and width of astrocytic stellar. ADK expressed in reactive astrocytes (Fig. 1F, G, arrows). The expression of ADK was prominent in perivascular areas (Fig. 1H, arrows). In addition, endothelial cells displayed marked ADK immunoreactivity (Fig. 1H, arrowheads) within the damaged hemosphere.

For the reason that the majority of the neuronal cells are mature cells with no ADK expression, these cells are regarded as the chief source for the direct release of adenosine . In RE, ADK was found not only expressed in reactive astrocytes, but also in a subpopulation of the remaining neuronal cells in cortical gray matter within the lesion area, with a predominant cytoplasmic local-ization (Fig. 1F, inset, arrowheads). Notably in a subset of lesional neurons representing 38.5%, 47.1%, and 56.7% of residual neurons in the mild, moderate, and severe RE, that may be part of the pathophysiology of RE or represent a revertant fetal expression pattern . The neuronal expression of ADK in the lesion of RE cortex maybe inflammation associated, indicating a potential additional layer of modulatory cross talk between the astrocyte-based adenosine cycle and inflammation. Another hypothesis on neuronal expres-sion of ADK in RE is that the early stage of develop-mental microenvironment alteration might destroy the transition of neuronal ADK from fetal to postnatal brains . Potential gene regulatory mechanisms includ-ing DNA promoter methylation, histone modifications and transcription factor binding may influence the dynamic regulation of the ADK gene during early postnatal brain development and maturation. Recentstudy indicated that binding of transcription factor specificity protein 1 to the ADK promoter influences the regulation of ADK expression .

Figure.1. Brain atrophy and overexpression of ADK in RE (A–C) The brain MRI (axial T2-weighted fluid attenuated inversion recovery image) of patient no. 1 displayed a typical progressive abnormal signal area expansion and atrophy of the left hemisphere (A, 1 months, B, 13 months, C, 15 months after the first seizure onset). (D, E) GFAP-positive reactive astrocytes were demonstrated in cortical gray matter (D, arrows) and white matter (E, arrows) within the lesion area. (F, G) Cytoplasmic localization of ADK immunoreactivity in cells with typical astroglial morphology (F, arrows) and a subpopulation of the remnant of neurons (F,arrowheads in inset) in cortical gray matter and white matter (G,arrows) within the lesion. (H) Cytoplasmic staining showed ADK in perivascular reactive astrocytes (arrows) and endothelial cells arrowheads within the lesions. Scale bars = (D-G) 50μm; (H) 12.5μm.

2. Overexpression of ADK in FCD

FCD is developmental malformation of the cerebral cortex that is highly associated with pharmacoresistant epilepsy in children and young adults. One of the commonest forms of FCD in children is FCD type IIB (FCDIIB) , which is characterized by cytomegalicdysmorphic neurons (Fig.2E, arrows) and a unique population of abnormal cells known as balloon cells (BCs) (Fig.2E, arrowheads) . Balloon cells are identi-fied as abnormal elements characterized by huge size, ill-defined membrane, pale cytoplasm and one or more eccentric nuclei. These cells are heterogeneous popula-tions that express cell surface markers for pluripotential stem cells and proteins for multipotent progenitors, or immature neurons/glia. It is regarded that dysfunction of proliferation caused by early embryonic insult plays an important role in the formation of balloon cells .Balloon cells in patients with FCDIIB are thought to originate from glioneuronal progenitor cells, strongly suggesting that defects of neuronal and glial specifica-tions are important in the histogenesis of FCDIIB .

Neuroimaging hallmarks include hyperintense T2-sig-naling (Fig.2A-C, arrows) and a ‘‘transmantle sign’’.Clinical electrophysiological recordings demonstraterelative specific interictal spike patterns .

Overexpression of the ADK concomitant with astrogli-osis within the lesions of FCDIIB has been demonstrat-ed in the recent study . Reactive astrocytes, character-ized by a hypertrophic morphology with larger soma and increased length and width of astrocytic stellar, were found in the lesion area (Fig.2F, arrows). Marked GFAP-positive reactive astrogliosis was often observed around BCs (Fig.2F, arrows) in the lesions of FCDIIB. 45% BCs were observed to be GFAP positive (Fig.2F,arrowheads). Cytoplasmic expression of ADK was found in reactive astrocytes (Fig.2G, black arrows), and a total of 77% of BCs were ADK positive (Fig.2G,white arrows), and BCs expressing the different cell markers expressing different degrees of ADK in FCDIIB specimens.

High levels of ADK expression in BCs and reactive astrocytes within the lesion of FCDIIB is thought to downregulate the adenosine level, which might result in lowering the threshold of seizures and leading to recur-rent seizures. On the other hand, ADK may play a key role to influence the proliferation function of neural progenitor cells . Radial glial cells in the ventricular zone are the source of BCs in patients with FCDIIB, which retain an embryonic phenotype . The expression of ADK in the BCs may suggest that these cells fail to mature fully and therefore contin-ue to express embryonic genes and proteins. These immature cells may lack some of the cellular machinery for migration. This may explain at least in part the localization of the BCs in the white matter and the gray/white matter junction (deep cortical layers), which are similar to the ADK expression pattern at early stages of corticogenesis . Accordingly, the presence of BCs may indicate that the early stage of insults during the prenatal/in utero period leads to the dysfunction of the neural stem cells in the ventricular zone area, impair the normal function such as prolifera-tion, maturation, migration, and terminal differentiation of neural stem cells . At last, potential gene regulatory mechanisms may also influence the dynamic regulation of the ADK gene during early postnatal brain development and maturation .

Of note, activation of the mammalian target of rapamycin (mTOR) signaling pathways is regarded to be associated with focal malformation of cortical development. Recent study indicted that the nuclear isoform of ADK might be associated with regulation of cell proliferation througha combination of epigenetic and additional adenosine receptor indepen-dent mechanisms, such as interaction with the mTOR pathway.

Figure.2. Imaging, pathologic features and overexpression of ADK of FCD IIB (A-C) Hyperintensity in coronal (A), sagittal (B) and axial (C) MR FLAIR image reveals the center of an FCDIIB lesion (arows) in the right frontal lobe. (D) Hypertrophic morphology of reactive astrocytes was shown in the cortex of the lesion (arrows). (E) Enlarged dysmorphic neurons with enlarged nuclei and abnormal intracytoplasmic Nissl aggregates (arrowheads) and balloon cells (arrows) are pathologic hallmarks of FCDIIB. (F) GFAP positive BCs (arrowheads) and reactive astrocytes surround BCs (arrows); (E) Cytoplasmic localization of ADK immunoreactivity in cells with typical astroglial morphology (arrows), balloon cells (white arrows) and enlarged dysmorphic neurons (arrowheads). Scale bars = (E-G) 50μm; (H) 12.5μm.

3. Overexpression of ADK in MTLE

MTLE is regarded as the most common form of epilepsy in adults, and the hallmark neuropathological features in patients with intractable MTLE is hippocampal sclerosis (HS). Neuroim- aging hallmarks include signal hyperin- tensity in T2 images and volume reduc-tion in the hippocampus (Fig.3A, B, arrows). Hippocampal specimens of pharmacoresistant MTLE patients that underwent epilepsy surgery for seizure control reveal the characteristic pattern of segmental neuronal cell loss and concomitant astrogliosis (Fig.3C, D, arrows). The marked pathologic char- acteristic of HS is astrogliosis. Accord- ing to the histological patterns of subfield neuronal loss and astrogliosis, HS is divided into 3 subtypes . Concomitant with astrogliosis within the hippocampus in MTLE, overex- pression of the astroglial ADK and residual neurons in HS has recently been reported in pharmacoresistant MTLE. ADK immunoreactivity in both HS and temporal cortex of MTLE patients was observed in cells with typical astroglial morphology (Fig.3E, F, arrows).

Figure.3. Imaging, pathologic features and overexpression of ADK of MTLE (A, B) Coronal (A, arrows) and axial (B, arrows) MR FLAIR image reveals signal hyperintensity in T2 images and volume reduction in the hippocampus. (C, D) Neuronal cell loss and concomitant astrogliosis are shown in hippocampus. (E, F) Cytoplasmic localization of ADK immunoreactivity in cells with typical astroglial morphology (arrows), Colocalization of GFAP and ADK are shown in inset. Scale bars = (C, E) 100μm; (D, F) 50μm.

4. Overexpression of ADK in SWS

SWS, is regarded as a congenital neurocutaneous disorder with disrupting capillary venous vessels in the leptomeninges of the brain and choroid. A port-wine stain ipsilateral to the vascular malforma- tion is often observed in the face of the patient with SWS (Fig.4A, arrows) . The recurrent seizures and the epilepsy associated comor-bidities such as cognition deficits are the main clinical presentation . The epilepsy is often pharmacoresistant when the seizures onset appears at younger age in patients with SWS . Neuroimaging hallmark is that Gadolinium-enhanced MRI and CT scan typically shows leptomeningeal enhancement in all cases (Fig.4B-D, arrows).

Leptomeningeal angiomatosis, neuronal loss and astrogliosis is often observed in neuropathological study .

On the one hand, the main clinical presen-tation is the recurrent seizures and cogni-tion decline, On the other hand, dysfunc-tion of adenosine system plays an import-ant role in the ictogenesis and epileptogen-esis as well as its comorbidities . Our ecent study demonstrated that overex-pression of ADK is involved in the patho-genesis of SWS. ADK expression increased in reactive astrocytes (Fig.4G, arrowheads; Fig.4H, arrows) (Fig.4I-L) concomitant with astrogliosis within the lesions in SWS. In addition, a subpopula-

tion of neuronal cells in the lesion area showed marked expression of ADK with a predominant cytoplasmic localization (Fig.4G, arrows). Overexpression of ADK in SWS, with a predominant cytoplasmic localization, leads to adenosine deficiency in the focus of the lesion and developed to be the epileptogenic zone. From the previ-ous data we can conclude that overexpres-sion of ADK is a solid biomarker for epileptic seizures and development of chronic epilepsy.

5. Overexpression of ADK and epileptogenesis

ADK, the main adenosine removing enzyme, linked the connection of reactive astrogliosis and neuronal dysfunction of epilepsy . Focal ADK-related seizures might be the substrate for subsequent seizure generalization and seizures spread-ing, and might be the beginning of the formation of epileptogenic zone . Overexpression in astrocytic ADK in reactive astrogliosis has be regarded to downregulate the adenosine level in the brain, which lower the seizures threshold and cause recurrent chron-ic seizures . However, little is known about the functional implication of neuronal ADK in human brain. ADK-tg mice, with overexpression of transgenic ADK throughout the brain, with particularly high levels in hippocampal pyramidal neurons had been demonstrated to be sufficient to down-regulate of the tissue concentration of adenosine , and consequent subclinical seizures, contributing mechanism for seizures generation in epilepsy . Therefore, overex-pression of the major adenosine removing enzyme ADK in reactive astrocytes and subpopulation of remaining neurons plays an crucial role in the epilepto-gensis of neurodegenerative diseases such as MTLE, RE, FCD and SWS .

Figure.4. Imaging, pathologic features and overexpression of ADK of SWS

(A) Port wine stain is shown in distribution of trigeminal nerve presented in the patient (arrows). (B-D) Gadoliniumenhanced T1-weighted image of MR showed enhancing leptomeningeal vessels in left hemisphere. (E-F) GFAP-positive reactive astrocytes were demonstrated in cortical gray matter (E, arrows) and white matter (F, arrows) within the lesion area. (G-H) Cytoplasmic localization of ADK immunoreactivity is shown in cells with typical astroglial morphology (G, arrowheads in inset) in cortical gray matter and white matter (H, arrows), and a subpopulation of the remnant of neurons (G, arrows in inset) in cortical gray matter within the lesion. (I-L) Co-localization of ADK (J, red) with GFAP (I, green) and DABI (K, green) confirms expression of ADK, with a predominant cytoplasmic expression, in astroglial cells (L, arrows). The cell with negative GFAP, positive DAPI and positive ADK could be the ADK positive neuron (L, arrowheads). Scale bars = (E-H) 50μm; (I-L) 12.5μm.

6. Overexpression of ADK and comorbidi-ties associated epilepsy

Currently, more and more experimental and clinical research demonstrated the bidirectional relation between epilepsy and associated comorbidities . Patients with epilepsy, especially with disabling epilepsy, clinically demonstrate not only recurrent seizures, cognition deficits and psychiatric symptoms such as depression and psychosis. Therefore, there might be common underlying mechanisms between epilepsy and comor-bidities associated with epilepsy. The comorbidities and structural brain alterations such as FCD, MTLE and tuber sclerosis are regarded as the predictor for the onset of seizures; and of importance, comorbidities usually suggested the efficacy of the current treatment for epilepsy with antiepileptic drugs, epilepsy resection are intractable . Recurrent disabling seizures usually lead to the reorganization of neural circuits and activities in the brain, and subsequent clinical comorbidity syndrome such as cognition deficits, depression and psychosis .

As a critical upstream regulator of complex homeostat- ic and metabolic networks, ADK has been demonstrat- ed to play a crucial role in the regulation of cognition process. ADK-tg mice, with transgenic overexpression of ADK in the brain, demonstrated a series of cognition deficits . ADK overexpression has been proved to lead to functional concomitant alterations in dopaminergic and glutamatergic functions. Thereby, Adk-tg mice displayed severe cognition deficits including reference memory, working memory, and associative learning .

In addition, brain inflammation has been proven as the underlying mechanism of several neuropsychiatric conditions. The recurrent epileptic seizures lead to the inflammation in the brain, may contribute to the neuro-psychiatric dysfunction . Furthermore, through action on the adenosine receptors, extracellular adenosine has potent anti-inflammatory functions . ADK dysfunction is involved in several pathologies, including epilepsy, epilepsy associated cognition deficits and inflamma-tion. For example, RE is the progressive inflammatory disorder with pharmacoresistant focal epilepsy and EPC and cognition deficits . In fact, in recent years, novel treatment strategies have already been developed that make use of the intracerebral transplantation of rodent and human cells that are ADK deficient and, thus, release adenosine, which demonstrated antiepilep-tic and neuroprotective properties . Targeting on down-regulation of the molecular biomarker ADK may provide the ideal treatment with the benefits of anti-in-flammation, anti-epilepsy as well as cognitive neuro-protectiion .

Conclusions

ADK provides important upstream regulation of adenosine-based homeostatic function of the brain and that this mechanism is necessary and permissive to synaptic actions of adenosine acting on multiple pathways. Overexpression of ADK, both neuronal and astroglial, plays an important role in the epileptogenesis and comorbidities associated with epilepsy in human chronic epilepsy such as RE, FCD, MTLE and SWS. ADK-regulating strategies thus represent innovative therapeutic opportunities to reconstruct network homeostasis in these multiple clinical conditions. The therapeutic strategy of adenosine augmentation should be considered and explored deeply, which may greatly improve the prognosis of FCD, MTLE or SWS patients with contraindication for surgical resection. In addition, dysregulation of ADK is a ubiquitous pathologic marker for epilepsy. Therefore, ADK is also regarded as a diagnostic biomarker for epilepsy. In the future, development of a PET tracer for ADK in humans may provide new frontier tools towards evaluation for the development of epilepsy or to measure the effectiveness of therapeutic interventions.

Abbreviations

RE: Rasmussen encephalitis; SWS: Sturge-Weber syndrome; ADK: adenosine kinase; MTLE: Mesial temporal lobe epilepsy; FCD: Focal cortical dysplasias.

Acknowledgment

The authors thank Professor Detlev Boison (RS Dow Neurobiology Laboratories, Legacy Research Institute, Portland, OR) for his kind and generous gift of ADK antibody. This Project was supported by the Grant from the BIBD-PXM2013_014226_07_000084, National Natural Science Foundation of China (81571275).

Competing interests

The authors declare that they have no competing inter- ests.