We study lipids in the nervous system

Project 1

Investigating mechanism and therapeutics for hereditary spastic paraplegia associated with SPTSSA mutation

  • A monoallelic de novo variant, SPTSSAT51I, has recently been shown to cause a complex spastic paraplegia (cHSP) in two children. SPTSSA encodes a subunit of serine palmitoyltransferase (SPT), the enzyme that catalyzes the rate-limiting step of sphingolipid (SL) synthesis. SLs, critical building blocks of the nervous system, are abundant within the lipid portion of myelin membranes1,2. While defects in lysosomal enzymes required for SL degradation have long been known to cause neurological disorders, more recently mutations in SL biosynthetic genes have been associated with neurological disease4-6.

    Our preliminary data show that the SPTSSAT51I mutant subunit renders SPT refractory to regulation by the ORMDLs, a family of three highly homologous proteins that are pivotal feedback regulators of SPT activity and thereby of SL homeostasis. We found elevated sphingolipids after the peak of myelination in SptssaT51I het mice, when ORM regulation normally dampens sphingolipid production. These elevated levels of SLs coincide with neurological phenotypes including hind limb clasping, leading to our hypothesis that SPTSSAT51I impedes ORMDL regulation of SPT and leads to unrestrained SPT activity, which negatively impacts myelination and causes a complex spastic paraplegia.

    1. whether early development of the nervous system, which requires high rates of de novo SL synthesis to supply critical components of myelin, is highly dependent on ORMDL regulation to ensure that SPT activity does not outpace conversion of potentially cytotoxic precursors (e.g., ceramides) of the myelin-enriched SLs.

    2. The possibility that ORMDL-mediated repression of SPT is also critical for transitioning to lower rates of SL synthesis following the peak of myelination will also be investigated.

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Figure 1. Sequencing data of patient-derived fibroblasts bearing heterozygous mutation in the CSF1R gene (c.2381 T>C; p.Ile794Thr).

Project 2

in vitro and ex vivo precision genome editing for the treatment of CSF1R-related disorder

  • Adult Onset Leukodystrophy with Neuroaxonal Spheroids and Pigmented Glia (ALSP) is a rare, neurodegenerative, autosomal dominant disease caused by loss-of-function mutations in the colony-stimulating factor 1 receptor (CSF1R) gene located at 5q32 chromosome. CSF1R is a tyrosine kinase receptor, essential for regulating the proliferation and activation of microglial cells. There are currently no disease-modifying therapies, and supportive treatment is used to control some of the ALSP symptoms.

    Employing nucleofection we managed to directly deliver plasmid of interest to the nucleus of the fibroblasts that still maintain their normal morphology and adhesion ability post-transfection. We are currently determining the most efficient CSF1R gene editing blueprint and optimal CBE-gRNA combinations will be used in the future to edit other cell types for therapeutic effect.

    1. whether early development of the nervous system, which requires high rates of de novo SL synthesis to supply critical components of myelin, is highly dependent on ORMDL regulation to ensure that SPT activity does not outpace conversion of potentially cytotoxic precursors (e.g., ceramides) of the myelin-enriched SLs.

    2. The possibility that ORMDL-mediated repression of SPT is also critical for transitioning to lower rates of SL synthesis following the peak of myelination will also be investigated.

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Project 3

AAV-mediated gene therapy for adrenoleukodystrophy

  • X-linked adrenoleukodystrophy (X-ALD) is a devastating neurological disorder caused by mutations in the ABCD1 gene that encodes a peroxisomal ATP-binding cassette transporter (ABCD1) responsible for transport of CoA-activated very long-chain fatty acids (VLCFA) into the peroxisome for degradation. We used recombinant adenoassociated virus serotype 9 (rAAV9) vector for delivery of the human ABCD1 gene (ABCD1) to mouse central nervous system (CNS). In vitro, efficient delivery of ABCD1 gene was achieved in primary mixed brain glial cells from Abcd1-/- mice as well as X-ALD patient fibroblasts.

    Importantly, human ABCD1 localized to the peroxisome, and AAV-ABCD1 transduction showed a dose-dependent effect in reducing VLCFA. In vivo, AAV9-ABCD1 was delivered to Abcd1-/- mouse CNS by either stereotactic intracerebroventricular (ICV) or intravenous (IV) injections. Astrocytes, microglia and neurons were the major target cell types following ICV injection, while IV injection also delivered to microvascular endothelial cells and oligodendrocytes. IV injection also yielded high transduction of the adrenal gland. Importantly, IV injection of AAV9-ABCD1 reduced VLCFA in mouse brain and spinal cord. We found that AAV9-mediated ABCD1 gene transfer is able to reach target cells in the nervous system and adrenal gland as well as reduce VLCFA in culture and a mouse model of X-ALD.

    1. whether early development of the nervous system, which requires high rates of de novo SL synthesis to supply critical components of myelin, is highly dependent on ORMDL regulation to ensure that SPT activity does not outpace conversion of potentially cytotoxic precursors (e.g., ceramides) of the myelin-enriched SLs.

    2. The possibility that ORMDL-mediated repression of SPT is also critical for transitioning to lower rates of SL synthesis following the peak of myelination will also be investigated.

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Project 4

Characterizing protein expression and motor dysfunction in a mouse model of HexB (Sandhoff’s Disease)

  • Sandhoff's disease is a rare monogenetic lysosomal storage disease due to mutations in HEX B. Our lab has a transgenic knockout HEX B mouse to model Sandhoff's disease. We are studying expression patterns on HEX B and HEX A in the murine central nervous system. Furthermore, the HEX B knockout mouse has a distinct phenotype where it develops dystonia at about 4 months. We are interested in doing behavioral and pathological analyses for this movement disorder.

    1. whether early development of the nervous system, which requires high rates of de novo SL synthesis to supply critical components of myelin, is highly dependent on ORMDL regulation to ensure that SPT activity does not outpace conversion of potentially cytotoxic precursors (e.g., ceramides) of the myelin-enriched SLs.

    2. The possibility that ORMDL-mediated repression of SPT is also critical for transitioning to lower rates of SL synthesis following the peak of myelination will also be investigated.

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Project 5

Piezo 2-mediated mechanosensitivity in a mouse model of AMN

  • Mutations in the peroxisomal half-transporter ABCD1 cause X-linked adrenoleukodystrophy, resulting in elevated very long-chain fatty acids (VLCFA), progressive neurodegeneration and an associated pain syndrome that is poorly understood. In the nervous system of mice, we found ABCD1 expression to be highest in dorsal root ganglia (DRG), with satellite glial cells (SGCs) displaying higher expression than neurons. We subsequently examined sensory behavior and DRG pathophysiology in mice deficient in ABCD1 compared to wild-type mice.

    Beginning at 8 months of age, Abcd1-/y mice developed persistent mechanical allodynia. DRG had a greater number of IB4-positive nociceptive neurons expressing PIEZO2, the mechanosensitive ion channel. Blocking PIEZO2 partially rescued the mechanical allodynia. Beyond affecting neurons, ABCD1 deficiency impacted SGCs, as demonstrated by high levels of VLCFA, increased glial fibrillary acidic protein (GFAP), as well as genes disrupting neuron-SGC connectivity. These findings suggest that lack of the peroxisomal half-transporter ABCD1 leads to PIEZO2-mediated mechanical allodynia as well as SGC dysfunction. Given the known supportive role of SGCs to neurons, this elucidates a novel mechanism underlying pain in X-linked adrenoleukodystrophy.

    1. whether early development of the nervous system, which requires high rates of de novo SL synthesis to supply critical components of myelin, is highly dependent on ORMDL regulation to ensure that SPT activity does not outpace conversion of potentially cytotoxic precursors (e.g., ceramides) of the myelin-enriched SLs.

    2. The possibility that ORMDL-mediated repression of SPT is also critical for transitioning to lower rates of SL synthesis following the peak of myelination will also be investigated.

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