Hereditary Spastic Paraplegia-Associated Mutations in theNIPA1Gene and ItsCaenorhabditis elegansHomolog Trigger Neural DegenerationIn VitroandIn Vivothrough a Gain-of-Function Mechanism

Abstract

We studied the consequences of expression of wild-type (WT) human NIPA1 and two mutant forms of NIPA1 with known HSP-associated mutations (T45R and G106R) on cultured rat cortical neurons and using equivalent substitutions in the Caenorhabditis elegans NIPA1 homolog CeNIPA. WT NIPA1 localized in transfected neuronal and non-neuronal cells to the Golgi complex, a subset of synaptic vesicles, to a subset of early endosomes, and plasma cell membrane. Mutant NIPA1 accumulated in the endoplasmic reticulum (ER) triggering ER stress and features of apoptotic cell death. Flow cytometric analysis of NIPA1 surface expression demonstrated relatively intact trafficking of mutant forms and only the T45R mutant exhibited modestly reduced patterns of surface expression without evidence for a dominant-negative effect. In vivo pan-neuronal expression of the WT C. elegans NIPA1 homolog (CeNIPA) was well tolerated, with no obvious impact on neuronal morphology or behavior. In striking contrast, expression of CeNIPA bearing HSP-associated mutations caused a progressive neural degeneration and a clear motor phenotype. Neuronal loss in these animals began at day 7 and by day 9 animals were completely paralyzed. These effects appeared to arise from activation of the apoptotic program triggered by unfolded protein response (UPR), as we observed marked modifications of motor and cellular phenotype when mutant NIPA1 was expressed in caspase (ced-3)-and UPR (xbp-1)-deficient backgrounds. We propose that HSP-associated mutations in NIPA1 lead to cellular and functional deficits through a gain-of-function mechanism supporting the ER accumulation of toxic NIPA1 proteins.

GABAA Receptor α1 Subunit Mutation A322D Associated with Autosomal Dominant Juvenile Myoclonic Epilepsy Reduces the Expression and Alters the Composition of Wild Type GABAA Receptors

“…Flow Cytometry and Fluorescent Resonance Energy Transfer (FRET)-Flow cytometry and FRET were performed as described previously (24,25). Briefly, transfected cells were detached from the plastic plates by incubating them with trypsin for 1 min at room temperature, a process that does not cause degradation of surface GABA A R protein (24).…”

“…We identified viable cells based upon their forward and side scatter properties (data not shown) that were originally determined from staining with the membrane-impermeable dye, 7-aminoactinomycin D (Invitrogen) (24,25). The A647 fluorophore was excited by a 635 nm laser and detected with a 675/20 bandpass filter, and the A555 fluorophore was excited with a 535 nm laser and detected with a 575/26 bandpass filter.…”

“…We extended these studies and used FRET to determine whether ␣1(AD) subunits specifically associated with wild type GABA A Rs. FRET measures the nonradiative transfer of energy between two fluorophores that are situated within at least 10 nm of one another and can be performed in situ in cells using flow cytometry (25,27).…”

Chemical Genetic Screens for TDP-43 Modifiers and ALS Drug Discovery

“…SPAS-1 is expressed in the cytoskeleton and is involved specifically in the disassembly of microtubules (Matsushita-Ishiodori et al, 2007). Then, the pan neuronal expression of NIPA-1 associated mutations led to motor deficits and shortened the lifespan of transgenic worms probably through the activation of caspases and increased ER toxicity (Zhao et al, 2008). With the rapid discovery of new HSP genes, more models are surely to come and will help unravel similarities between these diseases.…”

Genetic counseling for susceptibility loci and neurodevelopmental disorders: The del15q11.2 as an example

“…NIPA1 is an inhibitor of BMP (bone morphogenetic protein), important in axonal outgrowth, and it is thought that dysregulation of BMP lies at the basis of the HSP [Tsang et al, 2009;Botzolakis et al, 2011]. Unlike dominant negative mutations, there is no indication that a deletion of NIPA1, resulting in haploinsufficiency, would cause HSP [Rainier et al, 2003;Zhao et al, 2008].…”