Ilya M. Nasrallah

Targeted loss of Arx results in a developmental epilepsy mouse model and recapitulates the human phenotype in heterozygous females

Mutations in the X-linked aristaless-related homeobox gene (ARX) have been linked to structural brain anomalies as well as multiple neurocognitive deficits. The generation of Arx-deficient mice revealed several morphological anomalies, resembling those observed in patients and an interneuron migration defect but perinatal lethality precluded analyses of later phenotypes. Interestingly, many of the neurological phenotypes observed in patients with various ARX mutations can be attributed, in part, to interneuron dysfunction. To directly test this possibility, mice carrying a floxed Arx allele were generated and crossed to Dlx5/6(CRE-IRES-GFP)(Dlx5/6(CIG)) mice, conditionally deleting Arx from ganglionic eminence derived neurons including cortical interneurons. We now report that Arx(-/y);Dlx5/6(CIG) (male) mice exhibit a variety of seizure types beginning in early-life, including seizures that behaviourally and electroencephalographically resembles infantile spasms, and show evolution through development. Thus, this represents a new genetic model of a malignant form of paediatric epilepsy, with some characteristics resembling infantile spasms, caused by mutations in a known infantile spasms gene. Unexpectedly, approximately half of the female mice carrying a single mutant Arx allele (Arx(-/+);Dlx5/6(CIG)) also developed seizures. We also found that a subset of human female carriers have seizures and neurocognitive deficits. In summary, we have identified a previously unrecognized patient population with neurological deficits attributed to ARX mutations that are recapitulated in our mouse model. Furthermore, we show that perturbation of interneuron subpopulations is an important mechanism underling the pathogenesis of developmental epilepsy in both hemizygous males and carrier females. Given the frequency of ARX mutations in patients with infantile spasms and related disorders, our data unveil a new model for further understanding the pathogenesis of these disorders.

Identification of Arx transcriptional targets in the developing basal forebrain

Mutations in the aristaless-related homeobox (ARX) gene are associated with multiple neurologic disorders in humans. Studies in mice indicate Arx plays a role in neuronal progenitor proliferation and development of the cerebral cortex, thalamus, hippocampus, striatum, and olfactory bulbs. Specific defects associated with Arx loss of function include abnormal interneuron migration and subtype differentiation. How disruptions in ARX result in human disease and how loss of Arx in mice results in these phenotypes remains poorly understood. To gain insight into the biological functions of Arx, we performed a genome-wide expression screen to identify transcriptional changes within the subpallium in the absence of Arx. We have identified 84 genes whose expression was dysregulated in the absence of Arx. This population was enriched in genes involved in cell migration, axonal guidance, neurogenesis, and regulation of transcription and includes genes implicated in autism, epilepsy, and mental retardation; all features recognized in patients with ARX mutations. Additionally, we found Arx directly repressed three of the identified transcription factors: Lmo1, Ebf3 and Shox2. To further understand how the identified genes are involved in neural development, we used gene set enrichment algorithms to compare the Arx gene regulatory network (GRN) to the Dlx1/2 GRN and interneuron transcriptome. These analyses identified a subset of genes in the Arx GRN that are shared with that of the Dlx1/2 GRN and that are enriched in the interneuron transcriptome. These data indicate Arx plays multiple roles in forebrain development, both dependent and independent of Dlx1/2, and thus provides further insights into the understanding of the mechanisms underlying the pathology of mental retardation and epilepsy phenotypes resulting from ARX mutations.

Lis1 Is Necessary for Normal Non-Radial Migration of Inhibitory Interneurons

Type I lissencephaly is a central nervous system (CNS) malformation characterized by mental retardation and epilepsy. These clinical features suggest a deficit in inhibitory neurons may, in part, underlie the pathogenesis of this disorder. Mutations in, or deletions of, LIS1 are the most commonly recognized genetic anomaly associated with type I lissencephaly. The pathogenesis of type I lissencephaly is believed to be a defect in radial neuronal migration, a process requiring LIS1. In contrast the inhibitory neurons migrate non-radially from the basal forebrain to the neocortex and hippocampus. Given that Lis1 is expressed in all neurons, we hypothesized that Lis1 also functions in non-radial migrating inhibitory neurons. To test this hypothesis we used a combination of in vivo and in vitro studies with Lis1 mutant mice and found non-radial cell migration is also affected. Our data indicate Lis1 is required for normal non-radial neural migration and that the Lis1 requirement is primarily cell autonomous, although a small cell non-autonomous effect could not be excluded. These data indicate inhibitory neuron migration is slowed but not absent, similar to that found for radial cell migration. We propose that the defect in non-radial cell migration is likely to contribute to the clinical phenotype observed in individuals with a LIS1 mutation.

A polyalanine tract expansion in Arx forms intranuclear inclusions and results in increased cell death.

A growing number of human disorders have been associated with expansions of a tract of a single amino acid. Recently, polyalanine (polyA) tract expansions in the Aristaless-related homeobox (ARX) protein have been identified in a subset of patients with infantile spasms and mental retardation. How alanine expansions in ARX, or any other transcription factor, cause disease have not been determined. We generated a series of polyA expansions in Arx and expressed these in cell culture and brain slices. Transfection of these constructs results in nuclear protein aggregation, filamentous nuclear inclusions, and an increase in cell death. These inclusions are ubiquitinated and recruit Hsp70. Coexpressing Hsp70 decreases the percentage of cells with nuclear inclusions. Finally, we show that expressing mutant Arx in mouse brains results in neuronal nuclear inclusion formation. Our data suggest expansions in one of the ARX polyA tracts results in nuclear protein aggregation and an increase in cell death; likely underlying the pathogenesis of the associated infantile spasms and mental retardation.

Analysis of non-radial interneuron migration dynamics and its disruption in Lis1+/- mice.

Cell migration is an integral process in neural development. Analyses of radial cell migration (RCM) have revealed three modes of migration and specific defects in migration in various mouse mutants. In contrast, the dynamics of non‐radial cell migration (NRCM) are incompletely understood. To investigate the dynamics of NRCM, we utilized a slice culture assay coupled with time‐lapse videomicroscopy. This analysis revealed that non‐radially migrating cells have a complex pattern of extending and retracting one or multiple processes while the nucleus advances concurrently or independently. These data indicate that the process of interneuron migration is unique to that seen for any mode of RCM. Non‐radially migrating neurons moved for an average of 0.85 μm/min and paused for approximately 14% of the time observed. Given the novel morphology of NRCM, we hypothesized that specific aspects of migration would be defective with mutations in known cell migration genes, as described for RCM. This was tested by examining the dynamics of migration in the Lis1 mutant mouse; a well‐defined cell migration mutant with known defects in NRCM. In contrast to wild‐type cells, the rate of nuclear movement was significantly reduced in Lis1+/− interneurons, whereas the rate of active leading edge movement was similar. Morphologically, the leading process was significantly longer and the number of branches reduced in Lis1+/− mice. Together, these data indicate that the NRCM defect in Lis1+/− mice affects specific cellular processes. These data provide insight into NRCM and practical methods for future studies on the role(s) of specific genes in interneuron migration. J. Comp. Neurol. 496:847–858, 2006.

An overview of PET neuroimaging.

Over the past 35 years or so, PET brain imaging has allowed powerful and unique insights into brain function under normal conditions and in disease states. Initially, as PET instrumentation continued to develop, studies were focused on brain perfusion and glucose metabolism. This permitted refinement of brain imaging for important, non-oncologic clinical indications. The ability of PET to not only provide spatial localization of metabolic changes but also to accurately and consistently quantify their distribution proved valuable for applications in the clinical setting. Specifically, glucose metabolism brain imaging using (F-18) fluorodeoxyglucose continues to be invaluable for evaluating patients with intractable seizures for identifying seizure foci and operative planning. Cerebral glucose metabolism also contributes to diagnosis of neurodegenerative diseases that cause dementia. Alzheimer disease, dementia with Lewy bodies, and the several variants of frontotemporal lobar degeneration have differing typical patterns of hypometabolism. In Alzheimer disease, hypometabolism has furthermore been associated with poorer cognitive performance and ensuing cognitive and functional decline. As the field of radiochemistry evolved, novel radioligands including radiolabeled flumazenil, dopamine transporter ligands, nicotine receptor ligands, and others have allowed for further understanding of molecular changes in the brain associated with various diseases. Recently, PET brain imaging reached another milestone with the approval of (F-18) florbetapir imaging by the United States Federal Drug Administration for detection of amyloid plaque accumulation in brain, the major histopathologic hallmark of Alzheimer disease, and efforts have been made to define the clinical role of this imaging agent in the setting of the currently limited treatment options. Hopefully, this represents the first of many new radiopharmaceuticals that would allow improved diagnostic and prognostic information in these and other clinical applications, including Parkinson disease and traumatic brain injury.

Multimodal evaluation demonstrates in vivo 18 F-AV-1451 uptake in autopsy-confirmed corticobasal degeneration

in vivo evaluations of F-AV-1451 in patients with pathological confirmation. We report a multimodal evaluation of a 58-year-old male with autopsy-confirmed CBD. He participated in in vivo baseline (15 months pre-death) clinical, F-AV-1451 PET, F-florbetapir PET, MRI, and DTI and longitudinal (5 months pre-death) F-AV-1451 PET research studies (Online Resource 1). When enrolled in research, the patient met clinical criteria for progressive supranuclear palsy (Online Resource 2). Baseline F-AV-1451 (Fig. 1a) revealed the highest retention in deep grey matter areas commonly associated with CBD pathology [1], including bilateral substantia nigra, globus pallidus, and midbrain. Follow-up F-AV1451 revealed more visible retention in bilateral frontal and posterior temporal cortical regions along with midbrain and pons (Fig. 1b). A direct assessment of annualized change revealed a 1–9 % increase in F-AV-1451 retention, which was highest in the pons, medulla, and midbrain along with bilateral frontal and right temporo-parietal cortices (Fig. 1c). Baseline MRI (Fig. 2a) revealed predominantly reduced cortical grey matter in bilateral frontal cortex and right angular gyrus along with deep grey structures, including the midbrain, putamen, right globus pallidus, right caudate, and left hippocampus. White matter revealed increased mean diffusivity (a measure of white matter integrity) in the corpus callosum, bilateral tapetum, pontine crossing fibres, and right lateralized corticospinal tract, and posterior corona radiata. These observations are consistent with previously reported distributions of disease in CBD [6]. Spearman correlations revealed inverse associations between baseline F-AV-1451 and grey matter volume (rs = −0.209; p = 0.016; Fig. 2b) and mean diffusivity (rs = −0.329; p = 0.032; Fig. 2c). Furthermore, we Corticobasal degeneration (CBD) is characterized by 4-repeat misfolded tau (4Rtau) including astrocytic plaques, threads, and neuronal tangles [1]. F-AV-1451 is a PET radioligand that achieves in vivo binding in Alzheimer’s disease (AD) [8] and autoradiographic evidence of binding to paired helical filaments (PHFs) composed of 3-repeat misfolded tau (3Rtau) and 4Rtau characteristic of AD histopathology [4, 5, 7]. However, autoradiographic studies of F-AV-1451 on CBD postmortem tissue failed to demonstrate binding in cortical regions [5, 7], though there was minimal pathology in one study (<1.1 % tau-load) [7]. Another study suggests minimal, but present, autoradiographic binding of F-AV-1451 for 4Rtau [4]. Given mixed autoradiographic evidence in CBD, there is a need for

Multimodality Imaging of Alzheimer Disease and Other Neurodegenerative Dementias

Neurodegenerative diseases, such as Alzheimer disease, result in cognitive decline and dementia and are a leading cause of mortality in the growing elderly population. These progressive diseases typically have an insidious onset, with overlapping clinical features early in the disease course that make diagnosis challenging. The neurodegenerative diseases are associated with characteristic, although not completely understood, changes in the brain: abnormal protein deposition, synaptic dysfunction, neuronal injury, and neuronal death. Neuroimaging biomarkers—principally regional atrophy on structural MR imaging, patterns of hypometabolism on 18F-FDG PET, and detection of cerebral amyloid plaque on amyloid PET—are able to evaluate the patterns of these abnormalities in the brain to improve early diagnosis and help predict the disease course. These techniques have unique strengths and synergies in multimodality evaluation of the patient with cognitive decline or dementia. This review discusses the key imaging biomarkers from MR imaging, 18F-FDG PET, and amyloid PET; the imaging features of the most common neurodegenerative dementias; the role of various neuroimaging studies in differential diagnosis and prognosis; and some promising imaging techniques under development.

18F-flortaucipir tau positron emission tomography distinguishes established progressive supranuclear palsy from controls and Parkinson disease: A multicenter study

18F‐flortaucipir (formerly 18F‐AV1451 or 18F‐T807) binds to neurofibrillary tangles in Alzheimer disease, but tissue studies assessing binding to tau aggregates in progressive supranuclear palsy (PSP) have yielded mixed results. We compared in vivo 18F‐flortaucipir uptake in patients meeting clinical research criteria for PSP (n = 33) to normal controls (n = 46) and patients meeting criteria for Parkinson disease (PD; n = 26).

18F‐flortaucipir tau PET distinguishes established progressive supranuclear palsy from controls and Parkinson's disease: A multicenter study

18F‐flortaucipir (formerly 18F‐AV1451 or 18F‐T807) binds to neurofibrillary tangles in Alzheimer disease, but tissue studies assessing binding to tau aggregates in progressive supranuclear palsy (PSP) have yielded mixed results. We compared in vivo 18F‐flortaucipir uptake in patients meeting clinical research criteria for PSP (n = 33) to normal controls (n = 46) and patients meeting criteria for Parkinson disease (PD; n = 26).