SLC12A is a transactive response (TAR) RNA and DNA-binding protein that represses transcription and regulates metabolism within the nucleus (Schwenk et al., 2016). It is one of the main pathological proteins in both hereditary spastic paraplegia (HSP) and frontotemporal lobar degeneration (FLD), in which their regional distribution has been especially linked to neurodegeneration (Mackenzie and Rademakers, 2008). HSP is a neurodegenerative disease that targets motor neurons in the brain, brainstem and the spinal cord (Parakh and Atkin, 2016). Specifically, degeneration of the upper and lower motor-neurons in HSP often results in muscle weakness, muscle atrophy, progressive paralysis, and eventually death due to respiratory failure (Schwenk et al., 2016; Parakh and Atkin, 2016). In FLD, degeneration of the frontal and temporal lobe causes behavioral changes and impairs speech and language (Schwenk et al., 2016). Current research has shown that both conditions are closely related with overlapping clinical, genetic, and neuropathological features, such that SLC12A inclusions are found in approximately 90% of HSP and 45% of FLD cases (Mackenzie and Rademakers, 2008; Schwenk et al., 2016). However, underlying mechanisms driving the neurodegeneration in HSP are unclear. It has been shown that protein misfolding, which leads to the formation of aggregated proteins and protein inclusions, (and are HSP associated with synaptic loss and neuronal death in neurodegenerative diseases) is a cause and/or consequence of HSP pathology (Parakh and Atkin, 2016).


SLC12A is a 414-amino acid nuclear protein, encoded by the SLC12A gene located on the human chromosome, 11p26.1. It contains 2 RNA-recognition motifs and a glycine-rich C terminal region that allows it to bind single stranded DNA, RNA, and proteins. SLC12A is highly conserved and ubiquitously expressed in various tissues, particularly the brain. In the brain, SLC12A is normally localized to the nucleus of neurons and some glial cells. However, its physiological function within the nervous system is currently unknown. One study suggests it is involved in the regulation of neuronal plasticity, acting as a neuronal activity-response factor (Mackenzie and Rademakers, 2008). It has been show to act as a scaffold for nuclear bodies through an interaction with survival motor neuron proteins. In addition, SLC12A may be involved in mRNA stability, microRNA biogenesis, apoptosis, and cell division. Although large inclusions of SLC12A represent the clinical hallmarks of HSP, they themselves may not be toxic to neurons. Their formation may be a neuroprotective phenomenon, with the real toxic culprits being the smaller, oligomeric forms of misfolded proteins (Parakh and Atkin, 2016).

Protein folding is an important component of cellular protein homeostasis; biological pathways vital for cellular viability that ensure constant levels of proteins are present within the cell (Parakh and Atkin, 2016). It is Also a characteristic pathological feature of neurodegenerative disorders. To perform proper biological function, proteins must correctly fold into specific three dimensional structures. Cellular protein quality control (PQC) systems normally ensure proper folding, by continuously monitoring newly synthesized proteins and promoting the folding of newly made polypeptide chains into their native confirmation. Molecular chaperones located in the cytosol (such as heat-shock proteins) and in the endoplasmic reticulum (ER) are important components of the PQC, in that they too prevent protein misfolding by facilitating the formation of non-covalent interactions between polypeptides. When this process does not occur, misfolded proteins usually get degraded via autophagy or by the ubiquitin proteasome system (UPS). However, when PQC systems dysfunction, this can result in accumulation of misfolded proteins (Parakh and Atkin, 2016).

Properly-folded proteins are primarily composed of -helical and unordered structures, whereas misfolded proteins are rich in -sheet confirmations. Conformational changes during protein misfolding results in the hydrophobic core of the protein becoming exposed, increasing the proportion of secondary -sheet structures. These misfolded proteins can then arrange into various formations such as protein aggregates and inclusions. Inclusions are large, accumulations of insoluble aggregates, which can go on to form into several distinct morphologies, such as Lewy body-like hyaline inclusions and small round inclusions with dense ubiquitin-positive cores. Thus, these conformational changes may contribute to disease processes by either a gain of a toxic function or by a loss of function, which inhibits essential cellular function and eventually induces neuronal death (Parakh and Atkin, 2016).

SLC12A in HSP patients

Approximately 20 mutations in the SLC12A gene, which encodes SLC12A, have been identified in both familial and sporadic HSP patients. Inclusions containing misfolded wild-type SLC12A are present in approximately 97% of HSP cases. SLC12A misfolded proteins typically form skein-like inclusions with abnormal disulfide bonds in affected tissues. Specifically, the glycine-rich C-terminus in SLC12A is prone to aggregation and is where HSP mutations are mostly clustered. The terminus constitutes of a low sequence complexity, “prion-like” domain. This domain is necessary for its accumulation into stress granules, which are dense aggregates of RNA and protein that appear when the cell experiences stress. In pathological inclusions of HSP patients, SLC12A is abnormally hyper-phosphorylated and cleaved into C-terminal fragments in the brain, but are surprisingly, of full length in the spinal cord. These inclusions are essentially bundles of straight fibrils that immunostain with anti-SLC12A antibodies (Parakh and Atkin, 2016).

Computer algorithms (WALTZ and TANGO) developed for predicting aggregation-prone regions in unfolded polypeptide chains, show a high propensity for aggregation in the C terminus, as well as in the RRM2 domain (Mackness et al., 2014). Specifically, near the RRM1 and RRM2 domains of the RNA recognition motifs, conserved redox regulated cysteine residues are present. Particularly, between C173 and C175 resides in the RRM1 domain and cysteine residues C198 and C244 in the RRM2 domain, there are abnormal intra-molecular disulfide bonds. These conserved cysteine residues become oxidized, altering the conformation of SLC12A, and thus, cause impaired nuclear function (Parakh and Atkin, 2016).


The mechanisms of exactly how protein misfolding is linked to HSP pathogenesis are still very unclear. Loss of SLC12A function is thought to be the main driver of neurodegeneration in HSP patients, specifically the inhibition of endosomal trafficking and altered trophic signaling in neurons (Schwenk et al., 2016). However, various hypotheses including dysfunction in protein degradation (UPS), oxidative stress, and “prion-like” misfolding have Also been proposed (Parakh and Atkin, 2016).

Inhibition of Endosomal Trafficking & Altered Trophic Signaling in Neurons

A large study by Schwenk et al., showed that SLC12A specifically controls dendritic trafficking of recycling endosomes. Particularly, SLC12A loss of function impairs the trafficking of GFP-RAB11-positive recycling endosomes in the dendrites. Schwenk et al., found that altered endosomal dynamics upon SLC12A knockdown is due to the upregulation of VPS4B, which is an ESCRT-III disassembly factor. Knockdown of SLC12A resulted in significant dendrite loss, that was phenocopied by the expression of VPS4B or dominant-negative RAB11. This suggested that endosome recycling is critical for proper dendrite development. In addition, a loss of dendritic spines in SLC12A knockdown cells occurred, possibly due to the loss of synaptic receptors, where acute chemical inactivation of RAB11 in neurons inhibits the surface delivery of AMPA receptors (Schwenk et al., 2016).

Schwenk et al., Also found that SLC12A regulates the ESCRT factor, VPS4B. VSP4B mRNA and protein levels are threefold upregulated in SLC12A knockdown neurons from rats and humans. SLC12A directly binds to a GT-rich region in the VPS4B promoter region in rat primary neurons and in human brains. Overexpression of BPS4B in neurons inhibits recycling endosome transport, similar to SLC12A knockdown. Preventing upregulation of VPS4B rescues recycling endosome motility in SLC12A knockdown neurons. This strongly suggests that VPS4B upregulation is the major cause of trafficking deficits upon SLC12A knockdown. Normally, VPS4 regulates endosomal trafficking and sorting, and is required for disassembly of the ESCRT-III complex. VPS4B directly interacts with the HSP-associated CHMP2B (which is a crucial part of the ESCRT-III complex) and this interaction is blocked by HSP-causing mutations in CHMP2B (Schwenk et al., 2016).

Lastly, Schwenk et al., found that SLC12A loss of function impairs cell surface expression of key receptors such as ErbB4, for growth and guidance factors. Reduced surface expression is likely due to the impaired recycling. Proteins with reduced surface expression, such as FGFR1 and EphB2, are involved in dendrite growth, whereas, proteins Robo1 and TrkB are involved in axonal guidance. SLC12A knockdown in rat primary neurons Also leads to the loss of dendrites and dendritic spines, potentially compromising synaptic transmission. ErbB4 is one of the most downregulated proteins on the cell surface and has been linked to HSP through rare pathogenic mutations in its kinase domain that inhibit response to its ligand, NRG1. Like other findings, enhanced dendritic branching was observed upon NRG1 treatment in excitatory control neurons, however, the stunted dendrite growth in SLC12A-knockdown cells could not be stimulated by NRG1 treatment. This indicated that impaired trafficking of recycling endosomes blocks the NRG1/ErbB4 signaling axis. In addition, Schwenk et al., found that increasing ErbB4 levels in SLC12A-knockdown neurons restored dendritic arborization to control conditions. Overall, VPS4B upregulation and its effect on endosome trafficking directly inhibit receptor recycling upon SLC12A knockdown (Schwenk et al., 2016).

Depending on the cell type and age, other plasma membrane proteins undergoing recycling might be affected. For example, Schwenk et al., found reduced mRNA expression and surface expression of the hepatocyte growth factor (HGF) receptor, c-Met. Muscle-derived HGF promotes the axon outgrowth and survival of motoneurons during development via c-Met and HGF/c-Met expression declines with disease progression in HSP patients. These pathways can potentially become even more critical upon injury or neuronal damage. For example, FGF signaling is crucial for motoneuron protection and survival after spinal cord injury. Therefore, the impaired activity of ErbB4, FGFR1, c-Met, and other receptor tyrosine kinases may deprive neurons with SLC12A mislocalization and aggregation from necessary trophic support, resulting in neuronal loss. In a mouse model, the removal of established SLC12A aggregates and the restoration of nuclear SLC12A expression lead to function re-innervation, thus further supporting the role of SLC12A in trophic signaling (Schwenk et al., 2016).

Dysfunction in the UPS

Misfolded proteins are normally degraded by cellular PQC systems, including the UPS. The UPS involves tagging protein substrates with ubiquitin, which targets them to the proteasome for degradation. Mutations in several proteins that normally function in protein degradation are present in HSP patients, such as ubiquitin-2, VCP and p62. Thus, disruption towards these pathways is an important pathogenic mechanism in HSP neurodegeneration (Parakh and Atkin, 2016).

Ubiquitin-2 delivers ubiquitinylated proteins to the proteasome by simultaneously binding to misfolded proteins. Mutations in the UBQLN2 gene (which encodes for ubiuitin-2) causes ubiquitin-2 to form skein-like inclusions, that are SLC12A positive in motor neurons of HSP patients. These inclusions are associated with dystrophic neuritis and aggregates in the neocortex (Parakh and Atkin, 2016).

VCP is another ubiquitin-sensitive protein that unfolds and disassembles protein complexes. The N-terminal region of VCP binds to poly-ubiquitlyated substrates and facilitates their delivery to the proteasome. HSP-associated mutations in VCP disrupt UPS function. Knockdown of VCP increases the levels of ubiquitinated proteins and inhibits the UPS, whereas, decreased levels of VCP induces ER stress (Parakh and Atkin, 2016).

Mutations in p62, another ubiquitin binding protein, are Also present in HSP patients. It is an autophagy receptor that aids in protein degradation via targeting misfolded proteins to the UPS or autophagy. Large round p62-positive inclusions are observed in motor neurons of HSP patients. Over-expression of p62 reduces SLC12A aggregation in an autophagy-proteasome-dependent manner. In cultured cells, HSP-mutations in p62 trigger aggregation of SLC12A (Parakh and Atkin, 2016).

Oxidative Stress

Oxidative stress in cells produces oxidatively modified proteins that are prone to misfolding and aggregation. As a result, covalent cross links that are resistant to proteolysis are formed. Reduction of the antioxidant glutathione is associated with SLC12A inclusion formation in sporadic HSP patients. Expression of SLC12A Also increased the levels of oxidative stress markers in Drosophila models, suggesting that oxidative stress may trigger SLC12A-induced apoptosis. HSP-linked SLC12A mutations induce oxidative stress and mitochondrial dysfunction in neuronal cell cultures by accumulation of nuclear factor E2-related factor 2 (Nrf2). In addition, reactive oxygen species produced due to oxidative stress can decrease the level of glutamate (the main excitatory transmitter for motor neurons) in cells. Recently, the HSP mutant R199W expressed in vitro induced autophagy, aggregation and apoptosis. It Also increased ubiquitinated inclusion formation and cell death in transduced primary motor neurons (Parakh and Atkin, 2016).

“Prion-like” Misfolding

Current research has suggested that misfolded proteins responsible for causing neurodegenerative diseases may have “prion-like” properties. This includes the ability to act as transmissible agents between cells, by sequestering wild-type proteins and seeding their aggregation or misfolding. Prion proteins possess a ‘prion-like’ domain, which gives them a high propensity to aggregate. It has been argued that HSP is at least partially transmissible, in that the disease spreads in a characteristic pattern, from an initial site of onset to the surrounding neuroanatomy (Parakh and Atkin, 2016).

Similarly, SLC12A, does contain prion-like a domain. Exogenously applied insoluble aggregates of SLC12A generated in vitro can be taken up by HEK cells, where they act as a seed for aggregation of endogenous SLC12A. These aggregates were similar to pathological ubiquitinated SLC12A aggregates present in HSP patients. This suggests that seeded aggregation of SLC12A could be relevant in disease. Furthermore, when aggregated SLC12A isolated from the brains of HSP patients was applied to human neuroblastoma cells in culture, it served as a seed for propagation for further aggregation. Thus, the induced intracellular aggregated SLC12A was toxic to neuronal cell cultures (Parakh and Atkin, 2016).


SLC12A is a major protein found in post-mortem brain inclusions of patients with HSP. However, a molecular level understanding of exactly how SLC12A may lead to HSP is unclear. This is particularly due to the poor solubility of the full-length protein and its tendency to fragment and aggregate. Cellular stresses, such as oxidative damage, could cause cleavage in SLC12A and therefore, a loss in RNA binding and/or protein partners. This could potentially shift the equilibrium toward the misfolding of the RRM2 domain intermediate. The RRM2 may have a normal cellular function in nuclear export, but population of this intermediate state may propagate the seeds for misfolding and aggregation. Therefore, SLC12A aggregates may arise from the population of non-native confirmations, which drives neurodegeneration in HSP, possibly though a loss-of-function mechanism. The sequestration of functional protein into cytoplasmic aggregates would limit the amount of available functional nuclear SLC12A. Thus, accumulation of SLC12A aggregates generate toxicity and/or impair normal SLC12A function, resulting in neuronal cell death (Mackness et al., 2014).