Isolation and Culture of Dorsal Root Ganglia (DRG) from Rodents.

Preparations of peripheral sensory neurons from rodents are essential for studying the molecular mechanism of neuronal survival and physiology. Although, isolating and culturing these neurons proves difficult, often these preparations are contaminated with nonneuronal proliferating cells. Here, we describe an isolation method using a Percoll gradient and an antimitotic reagent to significantly reduce the nonneuronal cell contamination while maintaining the integrity of the rodent sensory dorsal root ganglia (DRG) neurons.

The lncRNA LOC102549805 (U1) modulates neurotoxicity of HIV-1 Tat protein.

HIV-1 Tat is a potent neurotoxic protein that is released by HIV-1 infected cells in the brain and perturbs neuronal homeostasis, causing a broad range of neurological disorders in people living with HIV-1. Furthermore, the effects of Tat have been addressed in numerous studies to investigate the molecular events associated with neuronal cells survival and death. Here, we discovered that exposure of rat primary neurons to Tat resulted in the up-regulation of an uncharacterized long non-coding RNA (lncRNA), LOC102549805 (lncRNA-U1). Our observations showed that increased expression of lncRNA-U1 in neurons disrupts bioenergetic pathways by dysregulating homeostasis of Ca2+, mitigating mitochondrial oxygen reduction, and decreasing ATP production, all of which point mitochondrial impairment in neurons via the Tat-mediated lncRNA-U1 induction. These changes were associated with imbalances in autophagy and apoptosis pathways. Additionally, this study showed the ability of Tat to modulate expression of the neuropeptide B/W receptor 1 (NPBWR1) gene via up-regulation of lncRNA-U1. Collectively, our results identified Tat-mediated lncRNA-U1 upregulation resulting in disruption of neuronal homeostasis.

Atrophy and Death of Nonpeptidergic and Peptidergic Nociceptive Neurons in SIV Infection.

HIV-associated sensory neuropathy is a common neurologic comorbidity of HIV infection and prevails in the post-antiretroviral therapy (ART) era. HIV infection drives pathologic changes in the dorsal root ganglia (DRG) through inflammation, altered metabolism, and neuronal dysfunction. Herein, we characterized specific neuronal populations in an SIV-infected macaque model with or without ART. DRG neuronal populations were identified by neurofilament H-chain 200, I-B4 isolectin (IB4), or tropomyosin receptor kinase A expression and assessed for cell body diameter, population size, apoptotic markers, and regeneration signaling. IB4+ and tropomyosin receptor kinase A-positive neurons showed a reduced cell body size (atrophy) and decreased population size (cell death) in the DRG of SIV-infected animals compared with uninfected animals. IB4+ nonpeptidergic neurons were less affected in the presence of ART. DRG neurons showed accumulation of cleaved caspase 3 (apoptosis) and nuclear-localized activating transcription factor 3 (regeneration) in SIV infection, which was significantly lower in uninfected animals and SIV-infected animals receiving ART. Nonpeptidergic neurons predominantly colocalized with cleaved caspase 3 staining. Nonpeptidergic and peptidergic neurons colocalized with nuclear-accumulated activating transcription factor 3, showing active regeneration in sensory neurons. These data suggest that nonpeptidergic and peptidergic neurons are susceptible to pathologic changes from SIV infection, and intervention with ART did not fully ameliorate damage to the DRG, specifically to peptidergic neurons.

SIV-Mediated Synaptic Dysfunction Is Associated with an Increase in Synapsin Site 1 Phosphorylation and Impaired PP2A Activity.

Although the reduction of viral loads in people with HIV undergoing combination antiretroviral therapy has mitigated AIDS-related symptoms, the prevalence of neurological impairments has remained unchanged. HIV-associated CNS dysfunction includes impairments in memory, attention, memory processing, and retrieval. Here, we show a significant site-specific increase in the phosphorylation of Syn I serine 9, site 1, in the frontal cortex lysates and synaptosome preparations of male rhesus macaques infected with simian immunodeficiency virus (SIV) but not in uninfected or SIV-infected antiretroviral therapy animals. Furthermore, we found that a lower protein phosphatase 2A (PP2A) activity, a phosphatase responsible for Syn I (S9) dephosphorylation, is primarily associated with the higher S9 phosphorylation in the frontal cortex of SIV-infected macaques. Comparison of brain sections confirmed higher Syn I (S9) in the frontal cortex and greater coexpression of Syn I and PP2A A subunit, which was observed as perinuclear aggregates in the somata of the frontal cortex of SIV-infected macaques. Synaptosomes from SIV-infected animals were physiologically tested using a synaptic vesicle endocytosis assay and FM4-64 dye showing a significantly higher baseline depolarization levels in synaptosomes of SIV+-infected than uninfected control or antiretroviral therapy animals. A PP2A-activating FDA-approved drug, FTY720, decreased the higher synaptosome depolarization in SIV-infected animals. Our results suggest that an impaired distribution and lower activity of serine/threonine phosphatases in the context of HIV infection may cause an indirect effect on the phosphorylation levels of essential proteins involving in synaptic transmission, supporting the occurrence of specific impairments in the synaptic activity during SIV infection.SIGNIFICANCE STATEMENT Even with antiretroviral therapy, neurocognitive deficits, including impairments in attention, memory processing, and retrieval, are still major concerns in people living with HIV. Here, we used the rhesus macaque simian immunodeficiency virus model with and without antiretroviral therapy to study the dynamics of phosphorylation of key amino acid residues of synapsin I, which critically impacts synaptic vesicle function. We found a significant increase in synapsin I phosphorylation at serine 9, which was driven by dysfunction of serine/threonine protein phosphatase 2A in the nerve terminals. Our results suggest that an impaired distribution and lower activity of serine/threonine phosphatases in the context of HIV infection may cause an indirect effect on the phosphorylation levels of essential proteins involved in synaptic transmission.

Perturbation of synapsins homeostasis through HIV-1 Tat-mediated suppression of BAG3 in primary neuronal cells.

HIV-1 Tat is known to be released by HIV infected non-neuronal cells in the brain, and after entering neurons, compromises brain homeostasis by impairing pro-survival pathways, thus contributing to the development of HIV-associated CNS disorders commonly observed in individuals living with HIV. Here, we demonstrate that synapsins, phosphoproteins that are predominantly expressed in neuronal cells and play a vital role in modulating neurotransmitter release at the pre-synaptic terminal, and neuronal differentiation become targets for Tat through autophagy and protein quality control pathways. We demonstrate that the presence of Tat in neurons results in downregulation of BAG3, a co-chaperone for heat shock proteins (Hsp70/Hsc70) that is implicated in protein quality control (PQC) processes by eliminating mis-folded and damaged proteins, and selective macroautophagy. Our results show that treatment of cells with Tat or suppression of BAG3 expression by siRNA in neuronal cells disturbs subcellular distribution of synapsins and synaptotagmin 1 (Syt1) leading to their accumulation in the neuronal soma and along axons in a punctate pattern, rather than being properly distributed at axon-terminals. Further, our results revealed that synapsins partially lost their stability and their removal via lysosomal autophagy was noticeably impaired in cells with low levels of BAG3. The observed impairment of lysosomal autophagy, under this condition, is likely caused by cells losing their ability to process LC3-I to LC3-II, in part due to a decrease in the ATG5 levels upon BAG3 knockdown. These observations ascribe a new function for BAG3 in controlling synaptic communications and illuminate a new downstream target for Tat to elicit its pathogenic effect in impacting neuronal cell function and behavior.

RNA-Based Therapy Utilizing Oculopharyngeal Muscular Dystrophy Transcript Knockdown and Replacement.

Oculopharyngeal muscular dystrophy (OPMD) is caused by a small expansion of a short polyalanine (polyAla) tract in the poly(A)-binding protein nuclear 1 protein (PABPN1). Despite the monogenic nature of OPMD, no treatment is currently available. Here we report an RNA replacement strategy that has therapeutic potential in cell and C. elegans OPMD models. We develop selective microRNAs (miRNAs) against PABPN1, and we report that miRNAs and our previously developed hammerhead ribozymes (hhRzs) are capable of reducing the expression of both the mRNA and protein levels of PABPN1 by as much as 90%. Since OPMD derives from a very small expansion of GCG within the polyAla tract, our hhRz and miRNA molecules cannot distinguish between the wild-type and mutant mRNAs of PABPN1. Therefore, we designed an optimized-codon wild-type PABPN1 (opt-PABPN1) that is resistant to cleavage by hhRzs and miRNAs. Co-expression of opt-PABPN1 with either our hhRzs or miRNAs restored the level of PABPN1, concomitantly with a reduction in expanded PABPN1-associated cell death in a stable C2C12 OPMD model. Interestingly, knockdown of the PABPN1 by selective hhRzs in the C. elegans OPMD model significantly improved the motility of the PABPN1-13Ala worms. Taken together, RNA replacement therapy represents an exciting approach for OPMD treatment.

Dysregulation of Neuronal Cholesterol Homeostasis upon Exposure to HIV-1 Tat and Cocaine Revealed by RNA-Sequencing.

HIV-1 Tat protein is released from HIV-1-infected cells and can enter non-permissive cells including neurons. Tat disrupts neuronal homeostasis and may contribute to the neuropathogenesis in people living with HIV (PLWH). The use of cocaine by PLWH exacerbates neuronal dysfunction. Here, we examined the mechanisms by which Tat and cocaine facilitate alterations in neuronal homeostatic processes. Bioinformatic interrogation of the results from RNA deep sequencing of rat hippocampal neurons exposed to Tat alone indicated the dysregulation of several genes involved in lipid and cholesterol metabolism. Following exposure to Tat and cocaine, the activation of cholesterol biosynthesis genes led to increased levels of free cholesterol and cholesteryl esters in rat neurons. Results from lipid metabolism arrays validated upregulation of several processes implicated in the biogenesis of ß-amyloid and Alzheimer's disease (AD), including sterol o-acyltransferase 1/acetyl-coenzyme A acyltransferase 1 (SOAT1/ACAT1), sortilin-related receptor L1 (SORL1) and low-density lipoprotein receptor-related protein 12 (LRP12). Further studies in Tat-treated primary neuronal cultures and brain tissues from HIV-1 transgenic mice as well as SIV-infected macaques confirmed elevated levels of SOAT1/ACAT 1 proteins. Our results offer novel insights into the molecular events involved in HIV and cocaine-mediated neuronal dysfunction that may also contribute to neuropathogenic events associated with the development of AD.

Caspase-1-associated immune activation in an accelerated SIV-infected rhesus macaque model.

In the antiretroviral therapy (ART) era, chronic HIV infection is primarily associated with chronic inflammation driving comorbidities such as cardiovascular disease and neurocognitive impairment. Caspase-1 activation in leukocytes has been documented in HIV infection; however, whether caspase-1 activation and the downstream pro-inflammatory cytokines interleukin-1beta (IL-1ß) and interleukin-18 (IL-18) contribute to chronic inflammation in HIV comorbidities remains undetermined. The relationship between the caspase-1 cascade and persistent inflammation in HIV has not been investigated. Here, we used an accelerated simian immunodeficiency virus (SIV)-infected rhesus macaque model with or without ART to investigate the dynamics of caspase-1 and immune cell activation before infection, 21 days post infection (dpi), and necropsy. Caspase-1, IL-18, IL-1ß, and immune markers were measured both in the circulation and lymphoid tissues. We found a significant increase in caspase-1 and IL-18 in SIV infection that positively correlated with inflammatory monocytes and negatively correlated with CD4+ T cell counts. ART attenuated these effects at necropsy in the circulation. Further, lymph nodes from SIV+ or SIV+ART animals had increased activation of caspase-1 and potential upstream priming of the NF-κB pathway, indicating that tissue-specific immune activation persists with ART. Together, these results shed light on the interconnectedness of the caspase-1 pathway and peripheral immune activation and further indicate that ART is not sufficient for suppressing inflammation. The caspase-1 pathway may provide novel therapeutic targets to improve HIV-associated comorbidities and health outcomes in the context of viral suppression.

Network analysis of hippocampal neurons by microelectrode array in the presence of HIV-1 Tat and cocaine.

HIV-associated neurocognitive disorders affecting greater than 30% of patients are caused by HIV-1 infection of the CNS, and in part, include neurotoxic effects of the viral transactivator of transcription, Tat protein. In addition to increasing the risk for becoming HIV infected, cocaine abuse enhances the neuropathogenic impacts of HIV-1. To investigate the outcome of Tat and cocaine interference in the hippocampal neuronal network, cross-rank-corrlation was employed to develop a systematic framework to assess hippocampal neurons behavior cultured on multielectrode arrays. Tat and cocaine differentially disturbed neuronal spiking rates, amplitude, synchronous activity, and oscillations within the hippocampal neuronal network via potentiation of inhibitory neurotransmission. The Tat-mediated impairment of neuronal spiking was reversible by removal of Tat, which restored neuronal activity. The presence of astrocytes co-cultured with neuronal networks diminished the effects of Tat and cocaine on neuron function suggesting a role for astrocytes in stabilizing neuronal behavior and increasing neuronal spontaneous activities such as bursting amplitude, frequency, and wave propagation rate. Taken together, our studies indicate that the HIV protein Tat and cocaine impair hippocampal neuronal network functioning and that the presence of astrocytes alleviates network dysfunction pointing to a newly discovered pathway through which ionic homeostasis is maintained by neuron-glial crosstalk in the CNS.

WNK Kinase Signaling in Ion Homeostasis and Human Disease.

WNK kinases, along with their upstream regulators (CUL3/KLHL3) and downstream targets (the SPAK/OSR1 kinases and the cation-Cl- cotransporters [CCCs]), comprise a signaling cascade essential for ion homeostasis in the kidney and nervous system. Recent work has furthered our understanding of the WNKs in epithelial transport, cell volume homeostasis, and GABA signaling, and uncovered novel roles for this pathway in immune cell function and cell proliferation.

KCC3 axonopathy: neuropathological features in the central and peripheral nervous system.

Hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum (HMSN/ACC) is an autosomal recessive disease of the central and peripheral nervous system that presents as early-onset polyneuropathy. Patients are hypotonic and areflexic from birth, with abnormal facial features and atrophic muscles. Progressive peripheral neuropathy eventually confines them to a wheelchair in the second decade of life, and death occurs by the fourth decade. We here define the neuropathologic features of the disease in autopsy tissues from eight cases. Both developmental and neurodegenerative features were found. Hypoplasia or absence of the major telencephalic commissures and a hypoplasia of corticospinal tracts to half the normal size, were the major neurodevelopmental defects we observed. Despite being a neurodegenerative disease, preservation of brain weight and a conspicuous absence of neuronal or glial cell death were signal features of this disease. Small tumor-like overgrowths of axons, termed axonomas, were found in the central and peripheral nervous system, indicating attempted axonal regeneration. We conclude that the neurodegenerative deficits in HMSN/ACC are primarily caused by an axonopathy superimposed upon abnormal development, affecting peripheral but also central nervous system axons, all ultimately because of a genetic defect in the axonal cotransporter KCC3.

Inhibition of HSV-1 Replication by Gene Editing Strategy.

HSV-1 induced illness affects greater than 85% of adults worldwide with no permanent curative therapy. We used RNA-guided CRISPR/Cas9 gene editing to specifically target for deletion of DNA sequences of the HSV-1 genome that span the region directing expression of ICP0, a key viral protein that stimulates HSV-1 gene expression and replication. We found that CRISPR/Cas9 introduced InDel mutations into exon 2 of the ICP0 gene profoundly reduced HSV-1 infectivity in permissive human cell culture models and protected permissive cells against HSV-1 infection. CRISPR/Cas9 mediated targeting ICP0 prevented HSV-1-induced disintegration of promonocytic leukemia (PML) nuclear bodies, an intracellular event critical to productive HSV-1 infection that is initiated by interaction of the ICP0 N-terminus with PML. Combined treatment of cells with CRISPR targeting ICP0 plus the immediate early viral proteins, ICP4 or ICP27, completely abrogated HSV-1 infection. We conclude that RNA-guided CRISPR/Cas9 can be used to develop a novel, specific and efficacious therapeutic and prophylactic platform for targeted viral genomic ablation to treat HSV-1 diseases.

Potassium-chloride cotransporter 3 interacts with Vav2 to synchronize the cell volume decrease response with cell protrusion dynamics.

Loss-of-function of the potassium-chloride cotransporter 3 (KCC3) causes hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), a severe neurodegenerative disease associated with defective midline crossing of commissural axons in the brain. Conversely, KCC3 over-expression in breast, ovarian and cervical cancer is associated with enhanced tumor cell malignancy and invasiveness. We identified a highly conserved proline-rich sequence within the C-terminus of the cotransporter which when mutated leads to loss of the KCC3-dependent regulatory volume decrease (RVD) response in Xenopus Laevis oocytes. Using SH3 domain arrays, we found that this poly-proline motif is a binding site for SH3-domain containing proteins in vitro. This approach identified the guanine nucleotide exchange factor (GEF) Vav2 as a candidate partner for KCC3. KCC3/Vav2 physical interaction was confirmed using GST-pull down assays and immuno-based experiments. In cultured cervical cancer cells, KCC3 co-localized with the active form of Vav2 in swelling-induced actin-rich protruding sites and within lamellipodia of spreading and migrating cells. These data provide evidence of a molecular and functional link between the potassium-chloride co-transporters and the Rho GTPase-dependent actin remodeling machinery in RVD, cell spreading and cell protrusion dynamics, thus providing new insights into KCC3's involvement in cancer cell malignancy and in corpus callosum agenesis in HMSN/ACC.

Comparative analysis of the expression profile of Wnk1 and Wnk1/Hsn2 splice variants in developing and adult mouse tissues.

The With No lysine (K) family of serine/threonine kinase (WNK) defines a small family of kinases with significant roles in ion homeostasis. WNK1 has been shown to have different isoforms due to what seems to be largely tissue specific splicing. Here, we used two distinct in situ hybridization riboprobes on developing and adult mouse tissues to make a comparative analysis of Wnk1 and its sensory associated splice isoform, Wnk1/Hsn2. The hybridization signals in developing mouse tissues, which were prepared at embryonic day e10.5 and e12.5, revealed a homogenous expression profile with both probes. At e15.5 and in the newborn mouse, the two probes revealed different expression profiles with prominent signals in nervous system tissues and also other tissues such as kidney, thymus and testis. In adult mouse tissues, the two expression profiles appeared even more restricted to the nervous tissues, kidney, thymus and testis, with no detectable signal in the other tissues. Throughout the nervous system, sensory tissues, as well as in Cornu Ammonis 1 (CA1), CA2 and CA3 areas of the hippocampus, were strongly labeled with both probes. Hybridization signals were also strongly detected in Schwann and supporting satellite cells. Our results show that the expression profiles of Wnk1 isoforms change during the development, and that the expression of the Wnk1 splice variant containing the Hsn2 exon is prominent during developing and in adult mouse tissues, suggesting its important role in the development and maintenance of the nervous system.

Loss of neuronal potassium/chloride cotransporter 3 (KCC3) is responsible for the degenerative phenotype in a conditional mouse model of hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum.

Disruption of the potassium/chloride cotransporter 3 (KCC3), encoded by the SLC12A6 gene, causes hereditary motor and sensory neuropathy associated with agenesis of the corpus callosum (HMSN/ACC), a neurodevelopmental and neurodegenerative disorder affecting both the peripheral nervous system and CNS. However, the precise role of KCC3 in the maintenance of ion homeostasis in the nervous system and the pathogenic mechanisms leading to HMSN/ACC remain unclear. We established two Slc12a6 Cre/LoxP transgenic mouse lines expressing C-terminal truncated KCC3 in either a neuron-specific or ubiquitous fashion. Our results suggest that neuronal KCC3 expression is crucial for axon volume control. We also demonstrate that the neuropathic features of HMSN/ACC are predominantly due to a neuronal KCC3 deficit, while the auditory impairment is due to loss of non-neuronal KCC3 expression. Furthermore, we demonstrate that KCC3 plays an essential role in inflammatory pain pathways. Finally, we observed hypoplasia of the corpus callosum in both mouse mutants and a marked decrease in axonal tracts serving the auditory cortex in only the general deletion mutant. Together, these results establish KCC3 as an important player in both central and peripheral nervous system maintenance.

Autocrine netrin function inhibits glioma cell motility and promotes focal adhesion formation.

Deregulation of mechanisms that control cell motility plays a key role in tumor progression by promoting tumor cell dissemination. Secreted netrins and their receptors, Deleted in Colorectal Cancer (DCC), neogenin, and the UNC5 homologues, regulate cell and axon migration, cell adhesion, and tissue morphogenesis. Netrin and netrin receptor expression have previously been shown to be disrupted in invasive tumors, including glioblastoma. We determined that the human glioblastoma cell lines U87, U343, and U373 all express neogenin, UNC5 homologues, and netrin-1 or netrin-3, but only U87 cells express DCC. Using transfilter migration assays, we demonstrate DCC-dependent chemoattractant migration of U87 cells up a gradient of netrin-1. In contrast, U343 and U373 cells, which do not express DCC, were neither attracted nor repelled. Ectopic expression of DCC by U343 and U373 cells resulted in these cells becoming competent to respond to a gradient of netrin-1 as a chemoattractant, and also slowed their rate of spontaneous migration. Here, in addition to netrins' well-characterized chemotropic activity, we demonstrate an autocrine function for netrin-1 and netrin-3 in U87 and U373 cells that slows migration. We provide evidence that netrins promote the maturation of focal complexes, structures associated with cell movement, into focal adhesions. Consistent with this, netrin, DCC, and UNC5 homologues were associated with focal adhesions, but not focal complexes. Disrupting netrin or DCC function did not alter cell proliferation or survival. Our findings provide evidence that DCC can slow cell migration, and that neogenin and UNC5 homologues are not sufficient to substitute for DCC function in these cells. Furthermore, we identify a role for netrins as autocrine inhibitors of cell motility that promote focal adhesion formation. These findings suggest that disruption of netrin signalling may disable a mechanism that normally restrains inappropriate cell migration.

KIF1A, an axonal transporter of synaptic vesicles, is mutated in hereditary sensory and autonomic neuropathy type 2.

Hereditary sensory and autonomic neuropathy type II (HSANII) is a rare autosomal-recessive disorder characterized by peripheral nerve degeneration resulting in a severe distal sensory loss. Although mutations in FAM134B and the HSN2 exon of WNK1 were associated with HSANII, the etiology of a substantial number of cases remains unexplained. In addition, the functions of WNK1/HSN2 and FAM134B and their role in the peripheral nervous system remain poorly understood. Using a yeast two-hybrid screen, we found that KIF1A, an axonal transporter of synaptic vesicles, interacts with the domain encoded by the HSN2 exon. In parallel to this screen, we performed genome-wide homozygosity mapping in a consanguineous Afghan family affected by HSANII and identified a unique region of homozygosity located on chromosome 2q37.3 and spanning the KIF1A gene locus. Sequencing of KIF1A in this family revealed a truncating mutation segregating with the disease phenotype. Subsequent sequencing of KIF1A in a series of 112 unrelated patients with features belonging to the clinical spectrum of ulcero-mutilating sensory neuropathies revealed truncating mutations in three additional families, thus indicating that mutations in KIF1A are a rare cause of HSANII. Similarly to WNK1 mutations, pathogenic mutations in KIF1A were almost exclusively restricted to an alternatively spliced exon. This study provides additional insights into the molecular pathogenesis of HSANII and highlights the potential biological relevance of alternative splicing in the peripheral sensory nervous system.

Transit defect of potassium-chloride Co-transporter 3 is a major pathogenic mechanism in hereditary motor and sensory neuropathy with agenesis of the corpus callosum.

Missense and protein-truncating mutations of the human potassium-chloride co-transporter 3 gene (KCC3) cause hereditary motor and sensory neuropathy with agenesis of the corpus callosum (HMSN/ACC), which is a severe neurodegenerative disease characterized by axonal dysfunction and neurodevelopmental defects. We previously reported that KCC3-truncating mutations disrupt brain-type creatine kinase-dependent activation of the co-transporter through the loss of its last 140 amino acids. Here, we report a novel and more distal HMSN/ACC-truncating mutation (3402C → T; R1134X) that eliminates only the last 17 residues of the protein. This small truncation disrupts the interaction with brain-type creatine kinase in mammalian cells but also affects plasma membrane localization of the mutant transporter. Although it is not truncated, the previously reported HMSN/ACC-causing 619C → T (R207C) missense mutation also leads to KCC3 loss of function in Xenopus oocyte flux assay. Immunodetection in Xenopus oocytes and in mammalian cultured cells revealed a decreased amount of R207C at the plasma membrane, with significant retention of the mutant proteins in the endoplasmic reticulum. In mammalian cells, curcumin partially corrected these mutant protein mislocalizations, with more protein reaching the plasma membrane. These findings suggest that mis-trafficking of mutant protein is an important pathophysiological feature of HMSN/ACC causative KCC3 mutations.