Seminars

Genome-wide association studies and functional genomics studies have linked specific cell types, genes, and pathways to Alzheimer’s disease (AD) risk. In particular, AD risk alleles primarily affect the abundance or structure, and thus the activity, of genes expressed in macrophages, strongly implicating microglia (the brain-resident macrophages) in the etiology of AD. These genes converge on pathways (endocytosis/phagocytosis, cholesterol metabolism, and immune response) with critical roles in core macrophage functions such as efferocytosis. Here, we review these pathways, highlighting relevant genes identified in the latest AD genetics and genomics studies, and describe how they may contribute to AD pathogenesis. Investigating the functional impact of AD-associated variants and genes in microglia is essential for elucidating disease risk mechanisms and developing effective therapeutic approaches." https://doi.org/10.1016/j.neuron.2022.10.015

Parkinson’s Disease (PD), the most common neurodegenerative movement disorder, is based on a complex interplay between genetic predispositions, aging processes, and environmental influences. In order to better understand the gene-environment axis in PD, we pursue a multi-omics approach to comprehensively interrogate genome-wide changes in histone modifications, DNA methylation, and hydroxymethylation, accompanied by transcriptomic profiling in cell and animal models of PD as well as large patient cohorts. Furthermore, we assess the plasticity of epigenomic modifications under influence of environmental factors using longitudinal cohorts of sporadic PD cases as well as mouse models exposed to specific environmental factors. Here, we present gene expression changes in PD mouse models in context of aging as well as environmental enrichment and high-fat diet.

The origins, mechanisms and consequences of epilepsy in the developing brain are incompletely understood. Many developmental epilepsies have a genetic basis and their mechanisms stem from deficits in the function of one or numerous genes. Others, such as those that follow prolonged febrile seizures or severe birth asphyxia in a ‘normal’ brain may depend on the interaction of the insult with the rapidly evolving brain cells and circuits. Yet, how early-life insults may provoke epilepsy is unclear, and requires multiple levels of analysis: behavior, circuits, cells [neurons, glia] and molecules. Here we discuss developmental epileptogenesis, addressing some of its special features: the epilepsy phenotype, the effects insults on the maturation of brain circuits, the role of neuron-glia-neuron communication in cellular and circuit refinement, and how transient epileptogenic insults provoke enduring changes in the structure, connectivity and function of salient neuronal populations. We will highlight resolved questions- and the many unresolved issues that require tackling in 2022 and beyond.

The Hsieh lab focuses on the mechanisms that promote neural stem cell self-renewal and differentiation in embryonic and adult brain. Using mouse models, video-EEG monitoring, viral techniques, and imaging/electrophysiological approaches, we elucidated many of the key transcriptional/epigenetic regulators of adult neurogenesis and showed aberrant new neuron integration in adult rodent hippocampus contribute to circuit disruption and seizure development. Building on this work, I will present our recent studies describing how GABA-mediated Ca2+ activity regulates the production of aberrant adult-born granule cells. In a new direction of my laboratory, we are using human induced pluripotent stem cells and brain organoid models as approaches to understand brain development and disease. Mutations in one gene, Aristaless-related homeobox (ARX), are of considerable interest since they are known to cause a common spectrum of neurodevelopmental disorders including epilepsy, autism, and intellectual disability. We have generated cortical and subpallial organoids from patients with poly-alanine expansion mutations in ARX. To understand the nature of ARX mutations in the organoid system, we are currently performing cellular, molecular, and physiological analyses. I will present these data to gain a comprehensive picture of the effect of ARX mutations in brain development. Since we do not understand how human brain development is affected by ARX mutations that contribute to epilepsy, we believe these studies will allow us to understand the mechanism of pathogenesis of ARX mutations, which has the potential to impact the diagnosis and care of patients.

Glymphatic perivascular exchange is supported by the astroglial water channel aquaporin-4 (AQP4), which localizes to perivascular astrocytic endfeet surrounding the cerebral vasculature. In aging mice, impairment of glymphatic function is associated with reduced perivascular AQP4 localization, yet whether these changes contribute to the development of neurodegenerative disease, such as Alzheimer’s disease (AD), remains unknown. Using post mortem human tissue, we evaluated perivascular AQP4 localization in the frontal cortical gray matter, white matter, and hippocampus of cognitively normal subjects and those with AD. Loss of perivascular and increasing cellular localization of AQP4 in the frontal gray matter was specifically associated with AD status, amyloid β (Aβ) and tau pathology, and cognitive decline in the early stages of disease. Using AAV-PHP.B to drive expression on non-perivascular AQP4 in wild type and Tg2576 (APPSwe, mouse model of Aβ deposition) mice, increased cellular AQP4 localization did not slow glymphatic function or change Aβ deposition. Using the Snta1 knockout line (which lacks perivascular AQP4 localization), we observed that loss AQP4 from perivascular endfeet slowed glymphatic function in wild type mice and accelerated Aβ plaque deposition in Tg2576 mice. These findings demonstrate that loss of perivascular AQP4 localization, and not increased cellular AQP4 localization, slows glymphatic function and promotes the development of AD pathology. To evaluate whether naturally occurring variation in the human AQP4 gene, or the alpha syntrophin (SNTA1), dystrobrevin (DTNA) or dystroglycan (DAG1) genes (whose products maintain perivascular AQP4 localization) confer risk for or protection from AD pathology or clinical progression, we evaluated 56 tag single nucleotide polymorphisms (SNPs) across these genes for association with CSF AD biomarkers, MRI measures of cortical and hippocampal atrophy, and longitudinal cognitive decline in the Alzheimer’s Disease Neuroimaging Initiative I (ADNI I) cohort. We identify 25 different significant associations between AQP4, SNTA1, DTNA, and DAG1 tag SNPs and phenotypic measures of AD pathology and progression. These findings provide complimentary human genetic evidence for the contribution of perivascular glymphatic dysfunction to the development of AD in human populations.

Large scale genetic studies and associated functional investigations have tremendously augmented our knowledge about the mechanisms underlying epileptic seizures, and sometimes also accompanying developmental problems. Pharmacotherapy of the epilepsies is routinely guided by trial and error, since predictors for a response to specific antiepileptic drugs are largely missing. The recent advances in the field of genetic epilepsies now offer an increasing amount of either well fitting established or new re-purposing therapies for genetic epilepsy syndromes based on understanding of the pathophysiological principles. Examples are provided by variants in ion channel or transporter encoding genes which cause a broad spectrum of epilepsy syndromes of variable severity and onset, (1) the ketogenic diet for glucose transporter defects of the blood-brain barrier, (2) Na+ channel blockers (e.g. carbamazepine) for gain-of-function Na+ channel mutations and avoidance of those drugs for loss-of-function mutations, and (3) specific K+ channel blockers for mutations with a gain-of-function defect in respective K+ channels. I will focus in my talk on the latter two including the underlying mechanisms, their relation to clinical phenotypes and possible therapeutic implications. In conclusion, genetic and mechanistic studies offer promising tools to predict therapeutic effects in rare epilepsies.

Diseases that arise from misfolding of an individual protein are rare. However, collectively, these folding diseases represent a large proportion of hereditary and acquired disorders. In fact, the term "Molecular Medicine" was coined by Linus Pauling in conjunction with the study of a folding disease, i.e. sickle cell anemia. In the past decade, we have witnessed an exponential growth in the number of mutations, which have been identified in genes encoding solute carriers (SLC). A sizable faction - presumably the majority - of these mutations result in misfolding of the encoded protein. While studying the export of the GABA transporter (SLC6A1) and of the serotonin transporter (SLC6A4), from the endoplasmic reticulum (ER), we discovered by serendipity that some ligands can correct the folding defect imparted by point mutations. These bind to the inward facing state. The most effective compound is noribogaine, the metabolite of ibogaine (an alkaloid first isolated from the shrub Tabernanthe iboga). There are 13 mutations in the human dopamine transporter (DAT, SLC6A3), which give rise to a syndrome of infantile Parkinsonism and dystonia. We capitalized on our insights to explore, if the disease-relevant mutant proteins were amenable to pharmacological correction. Drosopohila melanogaster, which lack the dopamine transporter, are hyperactive and sleepless (fumin in Japanese). Thus, mutated human DAT variants can be introduced into fumin flies. This allows for examining the effect of pharmacochaperones on delivery of DAT to the axonal territory and on restoring sleep. We explored the chemical space populated by variations of the ibogaine structure to identify an analogue (referred to as compound 9b), which was highly effective: compound 9b also restored folding in DAT variants, which were not amenable to rescue by noribogaine. Deficiencies in the human creatine transporter-1 (CrT1, SLC6A8) give rise to a syndrome of intellectual disability and seizures and accounts for 5% of genetically based intellectual disabilities in boys. Point mutations occur, in part, at positions, which are homologous to those of folding-deficient DAT variants. CrT1 lacks the rich pharmacology of monoamine transporters. Nevertheless, our insights are also applicable to rescuing some disease-related variants of CrT1. Finally, the question arises how one can address the folding problem. We propose a two-pronged approach: (i) analyzing the effect of mutations on the transport cycle by electrophysiological recordings; this allows for extracting information on the rates of conformational transitions. The underlying assumption posits that - even when remedied by pharmacochaperoning - folding-deficient mutants must differ in the conformational transitions associated with the transport cycle. (ii) analyzing the effect of mutations on the two components of protein stability, i.e. thermodynamic and kinetic stability. This is expected to provide a glimpse of the energy landscape, which governs the folding trajectory.

Alzheimer’s disease is the most common age-related neurodegenerative disorder. Familial forms of Alzheimer’s disease associated with the accumulation of a toxic form of amyloid-β (Aβ) peptides are linked to mitochondrial impairment. The coenzyme nicotinamide adenine dinucleotide (NAD+) is essential for both mitochondrial bioenergetics and nuclear DNA repair through NAD+-consuming poly (ADP-ribose) polymerases (PARPs). Here, we analysed the metabolomic changes in flies over-expressing Aβ and showed a decrease of metabolites associated with nicotinate and nicotinamide metabolism, which is critical for mitochondrial function in neurons. We show that increasing the bioavailability of NAD+ protects against Aβ toxicity. Pharmacological supplementation using NAM, a form of vitamin B that acts as a precursor for NAD+ or a genetic mutation of PARP rescues mitochondrial defects, protects neurons against degeneration and reduces behavioural impairments in a fly model of Alzheimer’s disease. Next, we looked at links between PARP polymorphisms and vitamin B intake in patients with Alzheimer’s disease. We show that polymorphisms in the human PARP1 gene or the intake of vitamin B, are associated with a decrease in the risk and severity of Alzheimer’s disease. We suggest that enhancing the availability of NAD+ by either vitamin B supplements or the inhibition of NAD+-dependent enzymes, such as PARPs are potential therapies for Alzheimer’s disease.

Dravet syndrome is a severe epileptic encephalopathy that begins during the first year of life and leads to severe cognitive and social interaction deficits. It is mostly caused by heterozygous loss-of-function mutations in the SCN1A gene, which encodes for the alpha-subunit of the voltage-gated sodium channel (Nav1.1) and is responsible mainly of GABAergic interneuron excitability. While different therapies based on the upregulation of the healthy allele of the gene are being developed, the dynamics of reversibility of the pathology are still unclear. In fact, whether and to which extent the pathology is reversible after symptom onset and if it is sufficient to ensure physiological levels of Scn1a during a specific critical period of time are open questions in the field and their answers are required for proper development of effective therapies. We generated a novel Scn1a conditional knock-in mouse model (Scn1aSTOP) in which the endogenous Scn1a gene is silenced by the insertion of a floxed STOP cassette in an intron of Scn1a gene; upon Cre recombinase expression, the STOP cassette is removed, and the mutant allele can be reconstituted as a functional Scn1a allele. In this model we can reactivate the expression of Scn1a exactly in the neuronal subtypes in which it is expressed and at its physiological level. Those aspects are crucial to obtain a final answer on the reversibility of DS after symptom onset. We exploited this model to demonstrate that global brain re-expression of the Scn1a gene when symptoms are already developed (P30) led to a complete rescue of both spontaneous and thermic inducible seizures and amelioration of behavioral abnormalities characteristic of this model. We also highlighted dramatic gene expression alterations associated with astrogliosis and inflammation that, accordingly, were rescued by Scn1a gene expression normalization at P30. Moreover, employing a conditional knock-out mouse model of DS we reported that ensuring physiological levels of Scn1a during the critical period of symptom appearance (until P30) is not sufficient to prevent the DS, conversely, mice start to die of SUDEP and develop spontaneous seizures. These results offer promising insights in the reversibility of DS and can help to accelerate therapeutic translation, providing important information on the timing for gene therapy delivery to Dravet patients.

Glioblastoma cells trigger pharmacoresistant seizures that may promote tumor growth and diminish the quality of remaining life. To define the relationship between growth of glial tumors and their neuronal microenvironment, and to identify genomic biomarkers and mechanisms that may point to better prognosis and treatment of drug resistant epilepsy in brain cancer, we are analyzing a new generation of genetically defined CRISPR/in utero electroporation inborn glioblastoma (GBM) tumor models engineered in mice. The molecular pathophysiology of glioblastoma cells and surrounding neurons and untransformed astrocytes are compared at serial stages of tumor development. Initial studies reveal that epileptiform EEG spiking is a very early and reliable preclinical signature of GBM expansion in these mice, followed by rapidly progressive seizures and death within weeks. FACS-sorted transcriptomic analysis of cortical astrocytes reveals the expansion of a subgroup enriched in pro-synaptogenic genes that may drive hyperexcitability, a novel mechanism of epileptogenesis. Using a prototypical GBM IUE model, we systematically define and correlate the earliest appearance of cortical hyperexcitability with progressive cortical tumor cell invasion, including spontaneous episodes of spreading cortical depolarization, innate inflammation, and xCT upregulation in the peritumoral microenvironment. Blocking this glutamate exporter reduces seizure load. We show that the host genome contributes to seizure risk by generating tumors in a monogenic deletion strain (MapT/tau -/-) that raises cortical seizure threshold. We also show that the tumor variant profile determines epilepsy risk. Our genetic dissection approach sets the stage to broadly explore the developmental biology of personalized tumor/host interactions in mice engineered with novel human tumor mutations in specified glial cell lineages.

Parkinson’s disease is affects millions of people around the world. The disease is characterized by typical movement defects that are caused by the loss of dopaminergic neurons, but several very debilitating non-motor symptoms occur more than 10 years before the motor symptoms. I will discuss how we study these non-motor symptoms including sleep disturbances and olfactory defects using large collections of knock in fruit flies that model the numerous familial forms of Parkinson’s disease as well as using human iPS cells from patients. A common emerging theme are defects in protein homeostasis that in specific neuronal cell types, cause cellular defects that explain the Parkinson-relevant phenotypes. Our work reveals the mechanisms that cause early defects in Parkinson’s disease and it opens therapeutic avenues to start tackling this disease.