I am a physician scientist conducting basic and translational research on pediatric epilepsy. In order to study epilepsy, my lab is organized around three major themes that have been shown through unbiased studies to be altered in epilepsy: 1) broad regulators of neuronal transcription 2) neural metabolism, and 3) neuronal morphology and cytoskeleton. By learning about rare genetic disorders within these domains and acquiring the thematic and technical expertise, we hope to develop new strategies for the treatment of common refractory epilepsy. This work is made possible by the collaborative environment at Brown University across the basic science department, Molecular Biology, Cell Biology, and Biochemistry and clinical departments including Neurology and Pediatrics.

Research Area 1: Transcriptional Mechanisms: Activity-dependent transcription has been highly studied with regard to normal neural function. An emerging theme is the coordinate regulation of many genes that are important for neuronal and synaptic function^10,11, including those important for neuronal metabolism^12,13 and those important for neuronal structure (cytoskeleton)¹⁴. Our work seeks to understand how this broad transcriptional regulation occurs in a disease context at different levels including epigenetic and transcription factor biology in both common causes of focal epilepsy and as a rare genetic disease. By studying both in the laboratory we can leverage shared expertise across projects.

           The circadian molecular clock is disrupted in human focal cortical dysplasia: My laboratory identified dysregulation in components of the molecular clock in epileptogenic tissue comprised of FCD cases andTSC-associated focal epilepsy¹⁵. We performed a functional study and found that deletion of CLOCK in excitatory neurons results in seizures.  These seizures are associated with non-REM sleep¹⁶. We have worked to understand how loss of the circadian molecular clock results in epilepsy¹⁵. We are working on downstream targets of CLOCK, the PARbZip transcription factors, which are also dysregulated in human FCD¹⁵. Mice with knockout of three structurally similar and functionally redundant PARbZip transcription factors have more severe spontaneous seizures than that of the CLOCK mice. We are performing an electrophysiological characterization of these triple PARbZip knockout mice and studying how these differ from CLOCK mice. The first study was published in Neuron¹⁵and I have been funded first by a CURE grant, then an R56 and subsequently by a current R01 from NINDS for this work. Luis Goicouria, a graduate student, and Haruki Higashimori work on this project in my laboratory.

         ASH1L haploinsufficiency causes highly penetrant autism and epilepsy: The ASH1L gene, encodes a histone methyl transferase and is a high-confidence Autism Spectrum Disorder (ASD) risk factor^17-19. From engaging with CARE4 ASH1L, the family organization, we realized that ASH1L patients have a high rate of epilepsy. Indeed, a large number of chromatin modifiers and transcription factors have been associated with ASD and epilepsy²⁰, and these may share mechanisms that impact neural development. We are characterizing a mouse model of ASH1L with respect to its epilepsy phenotype and electrophysiology.  Furthermore, we will test two therapeutic strategies for treating this disorder 1) balancing the counteracting epigenetic mechanism, PRC2²¹ and 2) re-expressing critical downstream targets of ASH1L. 

Research Area 2: Neural Metabolism: Metabolism refers to all of the chemical processes that sustain life; either by breaking down macromolecules to obtain energy to fuel cellular processes or the biosynthesis of macromolecules in order for cellular growth and maintenance. The complexity of metabolism is daunting; however, monogenic diseases offer powerful insights into mechanisms of metabolism in the human brain. Metabolism in epilepsy is critically important as evidenced by metabolic treatments such as the ketogenic diet.  However, this is understudied, and therapies for epilepsy targeting metabolism are not in widespread use.

           SLC13A5 infantile epileptic encephalopathy is a rare genetic disorder: The SLC13A5 Citrate Transporter Disorder is a newly-identified form of genetic epilepsy characterized by seizures beginning within the first days of life²⁵. As stated, the disease phenotype includes epilepsy, which starts in the neonatal period and can be difficult to control. Some patients have hundreds of seizures a day, which may be associated with cognitive impairment. Over time other effects become apparent including motor delays in standing/walking and problems with balance (ataxia). Impaired speech is observed in affected children with relative sparing of language comprehension. However, how mutations in SLC13A5 results in disease is presently unknown.

            The SLC13A5 gene encodes a plasma membrane citrate transporter which is highly expressed in brain and in liver. In the brain, SLC13A5 is expressed in both neurons and astrocytes ^26,27. Although citrate is generated and consumed in the mitochondria, cytoplasmic citrate is central to bio-energetic regulation. Cytoplasmic citrate is a potential source of the excitatory and inhibitory neurotransmitters, glutamate and GABA, respectively in neurons ^28,29. Thus, the normal function of SLC13A5 may be related to loss of function phenotypes in patients. This disorder is caused by bi-allelic mutations in the plasma membrane citrate transporter, SLC13A5, and thought to be autosomal recessive in nature^30,31.  In two of the most common mutations, the G219R mutation is thought to alter a sodium binding domain, and T227M is thought to alter citrate binding, affecting the normal function of the protein. The recurrence of specific missense mutations across unrelated families may indicate disease mechanisms more complex than loss of function alone. The work in animal models will guide the development of gene therapy for this rare genetic disorder. In addition to creating, characterizing, animal models, determining the genetic mechanism of this disease, I also perform clinical research on SLC13A5 citrate transporter disorder. I am the east coast site PI of a funded natural history characterizing the patients at Rhode Island Hospital/ Hasbro Children’s Hospital. This work in collaboration with the family group, the TESS foundation, is funded by a Rare as One Chan-Zuckerberg Awardee.

Research Area 3: Neuronal cytoskeleton is important for the formation and maintenance of neural connectivity. It is clear that the cytoskeleton is a key substrate for both correct development and maintenance of neural connectivity. Because the cytoskeleton is fundamental to development, mutations in genes encoding cytoskeleton proteins cause rare genetic disorders. Also, we and others find that cytoskeleton genes are differentially expressed in epileptogenic tissue that has undergone circuit reorganization. 

            A) Upregulation of Doublecortin-like kinase in human focal cortical dysplasia and during epileptogenesis: While DCX is the causative gene for X-linked lissencephaly, we find that its homologue doublecortin-like kinase (Dclk1) is upregulated in cases of human focal epilepsy. We investigated its role in the development of epilepsy using a mouse model of epilepsy, where severe continuous seizures (status epilepticus) are induced using a pharmacological agent, pilocarpine³⁵.  The mice recover and over the course of weeks develop spontaneous seizures. This model of temporal lobe epilepsy is associated with circuit changes in the hippocampus, including in the dentate gyrus where axons of granule neurons develop abnormal recurrent branches. These axons that normally project to the CA3 region of the hippocampus and the abnormal sprouting in epilepsy are thought to contribute to dysfunction of the dentate gyrus. Our ongoing work characterizing the role of DCLK1 in epilepsy shows that formation of abnormal branches in mossy fibers, or “mossy fiber sprouting” is greatly increased over baseline in mice with targeted deletions of DCLK1. We show that deletion of DCLK1 leads to increased epileptiform activity by EEG and abnormal increased activity in the structures downstream of the dentate gyrus by acute slice physiology. This work has been carried out by a current graduate student in the laboratory Brendan McCarthy-Sinclair and we are planning a publication for later in 2022 with a grant submission to follow in the next calendar year.

            B) X-linked lissencephaly caused by mutations in the microtubule binding protein Doublecortin: Regulation of the cytoskeleton in neuronal morphogenesis throughout development and beyond is critical for normal brain function. This is supported by human genetic data showing that cytoskeletal proteins, as a class, have been implicated in neural developmental disorders with intellectual disability and epilepsy. My work on doublecortin (DCX), encoded by a causative gene for lissencephaly, a human disorder resulting in epilepsy and intellectual disability, has resulted in our understanding of its role in neuronal development. We showed that DCX and DCX-family members are important not only during neuronal migration, but that they have significant roles in axon outgrowth and axon guidance³⁶. We showed that mice with targeted deletions of DCX and its structurally similar cognate protein, Doublecortin-like kinase 1 results in diminished or absent axon tracts³⁶.  The follow up study from my laboratory demonstrated that the axon defect included abnormalities in axon guidance³⁷.

I further defined the molecular mechanism of DCX family proteins and their interactions with other important cytoskeletal proteins. While microtubule structure Is largely unaffected by doublecortin family proteins, these proteins regulate the function of molecular motors. They inhibit the binding of conventional kinesin to microtubules. In contrast, they enhance binding and processivity of Kinesin-3 (also known as Kif1a), which is the motor that transports pre-synaptic vesicles in axons away from the neuronal cell body³⁸. To identify other motors regulated by DCX, we performed an in vivo proteomic screen³⁷ and identified two other motors selectively dysregulated in Dcx mutant neurons, the MT plus-end-directed motor, Kif21b, and the ubiquitous MT minus-end-directed motor, cytoplasmic dynein³⁷. In contrast to both kinesins, cytoplasmic dynein, which is essential for neuronal migration becomes highly associated with MTs in the absence of Dcx/Dclk1. Thus, Dcx appears to enhance kinesin-MT interactions while inhibiting dynein. These findings suggest the intriguing possibility that Dcx differentially regulates kinesin and dynein activities. We show that DCX changes the activity and composition of the dynein motor complex. In the absence of DCX, the adaptor protein, increases binding of the adaptor protein JIP3 to the dynein motor complex and dynein-mediated retrograde transport. In vitro studies to determine single molecule mediated motility of dynein with purified proteins from the laboratory of our collaborator, Arne Gennerich, Albert Einstein College of Medicine, demonstrates that these interactions are necessary and sufficient to regulate dynein. Based on these findings, we conclude that DCX inhibits dynein-mediated retrograde transport by inhibiting the association of JIP3 with dynein.

Altogether these studies represent functional studies on the lissencephaly interactome, with mutations in genes that encode the aforementioned proteins causing lissencephaly or related neural developmental disorders. Furthermore, they demonstrate the role of these “disease genes” in fundamental stages in neural development. We are submitting a publication and are planning a multi-PI grant submission in the next year based on these findings.

NIH/ NINDS R01 NS131865-01 “Genetic and Functional Mechanisms in Citrate Transporter Disorder associated with SLC13A5” 6/01/2023- 5/30/2028 

(Role: Contact PI, MPI)

TESS foundation/ Chan-Zuckerberg Rare as One Awardee: “Natural History Study of SLC13A5”

01/01/2022-12/31/2024 (100,000)

(East-coast site PI at Rhode Island Hospital)

1R01MH127081-01A1: Ash1l-mediated transcriptional networks in autism spectrum disorders

04/01/2022-03/31/2027 (825,000)

(MPI, with Sofia Lizarraga as contact PI)

RO1 NS104428-01: The circadian molecular clock in refractory focal epilepsy

NIH/ NINDS 2/01/2019-1/31/2023 (1,008,000)

(Role: PI)

2011                                        Epilepsy Foundation Grant

2013                                        NARSAD Young Investigator Award

2013                                        Whitehall Foundation Award

2017                                        CURE Sleep and Epilepsy Award

Department of Neurology,

Molecular Biology, Cell Biology, and Biochemistry

BIBS