Amplification of the HSR Improves the ALS Phenotype of mVCP Mice

The loss of functional motor units is the defining characteristic of ALS and accounts for the loss of innervation at the neuromuscular junction and the resulting muscle paralysis. We have previously reported that mice expressing the human transgene for mutant VCP (A232E) have significant muscle pathology and reduced muscle strength [33]. As shown in Fig. 1, electrophysiological analysis of 10–13 mice per experimental group of the same mVCP A232E strain revealed that these mice at 14 months of age also manifest significant motor deficits, reminiscent of ALS [27]. Thus, compared to control mice expressing wild-type human VCP (wtVCP), there was a 26.5% (p=0.0002) reduction in the number of functional motor units innervating the hindlimb extensor digitorum longus (EDL) muscle in mVCP mice (Fig. 1A, B), decreasing from an average of 34 ± 1.5 motor units in wtVCP mice to 25 ± 2 in mVCP mice.

Fig. 1

Loss of motor neurons and motor units in mVCP mice is improved with arimoclomol treatment. (A) Examples of isometric twitch force traces of the EDL muscle from representative non-transgenic wildtype (WT) control and mVCP mice. Each increment represents recruitment of a motor unit with increasing nerve stimulation. (B) Bar chart shows quantification of motor unit number in all experimental groups at 14 months of age. *** p=0.0002 (one-way ANOVA, n=10 WT, n=10 wtVCP, n=13 mVCP, n=10 mVCP+ arimoclomol mice per group, both hindlimbs assessed). (C) Nissl-stained images of spinal cord sections from the L4 region of wtVCP, mVCP and arimoclomol-treated mVCP mice at 14 months of age. Sciatic pool neurons are circled. Insets show higher magnification images. Scale bars = 20μm. (D) Bar chart showing number of motor neurons present in the spinal cord sciatic pool from a means of all animals per experimental groups. *p=0.029, ***p=0.0001 (one-way ANOVA, n=5 animals per group). (E) Bar chart representing the mean motor neuron area across cohorts. ***p=0.0001 (one-way ANOVA, n=3 animals per group). (F) Size distribution graph by total somal area (μm2) of sciatic pool motor neurons from WT, mVCP and arimoclomol-treated mVCP mice (from 10 images of spinal cord regions L4 and L5, one-way ANOVA, n=3 mice per group)

Amplification of the HSR in mVCP mice by daily treatment with 120mg/kg of arimoclomol, from 4 months (symptom onset) to 14 months of age, resulted in a complete prevention of motor unit loss in EDL muscles. In the arimoclomol-treated mVCP cohort, 35 ± 1.9 motor units innervated the EDL muscle (p=0.0002), which is similar to the number in both wtVCP mice (34 ± 1.5) and WT non-transgenic littermate controls (34.3 ± 0.9; Fig. 1B). There was no significant difference in the number of EDL motor units between the two control groups wtVCP and non-transgenic WT littermates.

Following these acute physiological experiments, the spinal cord and brain were removed for histopathological analysis. Quantification of the mean number of motor neurons in the sciatic motor pool, which innervate hind limb muscles, showed no significant difference in the number of motor neurons in wtVCP and non-transgenic WT controls, in which there were 725 ± 20 and 692 ± 20 motor neurons, respectively. However, there was a significant decrease in motor neuron survival in mVCP mice, in which only 494 ± 14 motor neurons survived; this represents a 32% reduction in motor neuron survival compared to wtVCP controls (p=0.0001, Fig. 1C, D). Amplification of the HSR by treatment with arimoclomol significantly improved motor neuron survival in mVCP mice, and 612 ± 42 motor neurons survived, an improvement of 24% (p=0.029) when compared to untreated mVCP mice.

Furthermore, in untreated mVCP mice, motor neurons, which survived at 14 months, had an abnormal morphology, with small, compacted cell bodies, contrasting with the large polygonal shape of motor neurons observed in control animals (Fig. 1C). We therefore assessed the total soma area of motor neurons in the sciatic pool in each cohort of mice (n= 3 per group). As can be seen in Fig. 1E, there was a clear reduction in the mean soma size of the motor neurons that survived in mVCP mice compared to wtVCP controls. Size distribution analysis revealed that the reduction in mean motor neuron soma area in mVCP mice was predominantly due to the preferential loss of large, likely alpha motor neurons, and an increase in the proportion of smaller motor neurons (Fig. 1F). This shift in the size distribution of motor neuron soma size in mVCP mice was prevented in arimoclomol-treated mVCP mice, in which the motor neuron soma size distribution was similar to that observed in normal WT mice (Fig. 1F).

Aggregation of misfolded proteins in ubiquitin-containing inclusions and cytoplasmic mislocalisation of the nuclear RNA-binding protein TDP-43 are key hallmarks of ALS pathology. We observed no ubiquitin pathology in the spinal cord of wtVCP mice or non-transgenic WT littermates at 14 months of age. In contrast, ubiquitin-positive protein aggregates were detected in motor neurons of mVCP mice (Fig. 2A). Whilst immunostaining for cytoplasmic TDP-43 was low in wtVCP tissue, a distinct increase in cytoplasmic TDP-43 was observed in motor neurons of mVCP mice (Fig. 2B). In contrast, in mVCP mice treated with arimoclomol, cytoplasmic TDP-43 mislocalisation and ubiquitin pathology were similar to that seen in control animals (Fig. 2B).

Fig. 2

Ubiquitin and TDP-43 pathology in mVCP mouse spinal cord is improved with arimoclomol and is associated with increased HSP70. Immunofluorescent images of lumber spinal cord sections from wtVCP, mVCP and arimoclomol-treated mVCP mice showing, (A) ubiquitin immunoreactivity in neurons (arrows indicate ubiquitin-positive protein aggregates), (B) TDP-43 localisation in neurons and, (C) HSP70 expression in spinal cord sections with and without neuronal marker (β-III tubulin, red). Nuclei labelled with DAPI (blue). (D) Bar graph representing the corrected total cell fluorescence intensities (CTCF) of HSP70 in immunolabelled spinal cord ventral horn motor neurons from each experimental group (average 216 neurons per group, one-way ANOVA, **p=0.0008, ***p= 0.0001). (E) Example image of HSP70 expression in GFAP co-labelled spinal cord sections with and without the glial marker from an arimoclomol-treated animal. White arrows show GFAP-negative neuronal cells; yellow arrows show GFAP-positive glial cells. DAPI labels nuclei (blue). Scale bar = 10μm

HSP70 levels in spinal motor neurons cord were quantified to better understand the HSR response in these cells directly. The pathological changes observed in the spinal cord of mVCP mice were associated with an almost 3-fold increase in the expression of HSP70 in motor neurons, compared to that in wtVCP control animals in which there was no significant pathology (Fig. 2C, D). Importantly, this indicates that the endogenous response to cell stress has been triggered in the mVCP motor neurons, although this was not sufficient to prevent the development of pathology. Amplification of the HSR in mVCP mice by treatment with arimoclomol resulted in approximately a 4-fold increase in HSP70 compared to controls (Fig. 2C, D). In addition, in arimoclomol-treated mVCP mice, HSP70 levels were also enhanced in non-neuronal cells, initially seen as filamentous-like structures negative for β-III tubulin (Fig. 2C), which subsequent GFAP-labelling confirmed to be astroglia (Fig. 2E). This likely reflects an additional cytoprotective response to mVCP-induced stress in the spinal cord.

VCP is known to play an essential role in autophagy [13], and dysfunctional autophagy has been implicated in the pathogenesis of ALS and MSP. For example, MSP patients expressing mVCP show evidence of disrupted autophagy, with accumulation of the key autophagic markers p62 (Sequestosome 1) and LC3 within myofibres [13, 38]. We therefore examined the pattern of expression of these two autophagic markers in the spinal cord of mVCP mice at 14 months of age. We observed an increase in p62 expression in both the white and grey matter of the spinal cord compared to control animals (Fig. 3A–C), with p62 aggregates clearly visible in motor neurons (Fig. 3A—magnified inset in mVCP image). Co-labelling with fluoromyelin suggested that the intense p62 staining observed in mVCP spinal cord sections was associated with oligodendrocytes (Fig. 3B). Closer examination of the p62-positive oligodendrocytes revealed gross myelin disruption around axons, deviating from the classic ‘onion bulb’ structure of healthy myelin, suggestive of axonal and/or myelin degeneration (Fig. 3C). This pattern of p62 expression was not observed in spinal cords from any control animals and was visibly reduced in arimoclomol-treated animals.

Fig. 3

Increased expression of p62 and LC3 in the spinal cord grey and white matter of mVCP mice is reduced with arimoclomol treatment. (A) Representative histological images of p62 expression in the spinal cord of wtVCP, mVCP and arimoclomol-treated mVCP mice. mVCP spinal cord shows increased p62 expression in white and grey matter and aggregated p62 in sciatic pool motor neurons (inset, high magnification image) and oligodendrocytes (ringed in red). (B) High magnification image of increased p62 expression with myelin shown in red. (C) High magnification image of collapsed myelin sheath in mVCP spinal cord white matter. (D) Immunohistochemistry showing LC3 expression in mouse spinal cord from wtVCP, mVCP and mVCP mice treated with arimoclomol, co-localised with myelin (red). Inset shows higher magnification image of an axon surrounded by myelin (red). Scale bar = 10μm

We also examined the expression pattern of the autophagosome marker LC3. LC3 is recruited to the autophagosomal membrane during autophagy and later degraded in the autolysosomal lumen and, as such, is routinely used as a marker of autophagic activity in cells [39, 40]. In mVCP mice we observed an increase in the expression of LC3 in the spinal cord and in particular in oligodendrocytes associated with abnormal myelination (Fig. 3D). Together with the accumulation of p62, this provides further evidence of defective autophagy in these cells due to the presence of mutant VCP. Importantly, the accumulation of p62 and LC3 and abnormal myelination was visibly reduced in mVCP mice in which the HSR was amplified by arimoclomol treatment.

Amplification of the HSR Improves the FTD Phenotype in the Brain of mVCP Mice

A third of patients diagnosed with MSP caused by mutations in VCP develop FTD [9], and mutations in VCP cause <1% of all FTD cases [41]. To determine whether mVCP mice exhibit FTD-like pathology, we next examined their brain.

Similar to ALS and MSP, cytoplasmic mislocalisation of TDP-43 is a pathological characteristic of FTD, with mislocalised TDP-43 present in the brain of approximately 50% of FTD cases, and brain pathology often indistinguishable from that seen in FTD patients with a mutation in the TARDBP gene itself [9, 42]. Cytoplasmic TDP-43 is often found either dispersed in the cytosol or within inclusion bodies, concomitant with its nuclear clearance [43], indicating that a loss of normal nuclear function as well as gain of toxic cytoplasmic function may play a role in disease pathogenesis [42]. In the brain of 14-month-old mVCP mice, we observed an increase in the number of cortical neurons with distinct cytoplasmic TDP-43 mislocalisation compared to control mice, with many neurons showing nuclear clearance of TDP-43 (Fig. 4A). In contrast, TDP-43 expression in the cortex of arimoclomol-treated mVCP mice was similar to control animals, and no cytoplasmic mislocalisation associated with nuclear clearance of TDP-43 was apparent in the brains of the arimoclomol-treated group (Fig. 4A).

Fig. 4

TDP and ubiquitin pathology in mVCP mouse brain is improved with arimoclomol treatment. (A) TDP-43 localisation in cortical cells. Insets show cells at higher magnification. Scale bar = 20μm. (B) Ubiquitin immunoreactivity in mouse cortex shows ubiquitin-positive aggregates in mVCP brain (red). Scale bar = 20μm. (C) Stress granule marker Tia1 colocalised with TDP-43 and ubiquitin in mVCP brain. Scale bar = 10μm. DAPI labels nuclei (blue) in all images

We next examined the expression of ubiquitin in the brain of 14-month old mVCP mice. Similar to our findings in the spinal cord, in mVCP mice we observed ubiquitin-positive intracellular aggregates in the brain, although these were not limited to the cortex. No ubiquitin-positive aggregates were detected in control animals, or in mVCP mice treated with arimoclomol (Fig. 4B).

Microtubule-associated protein tau (MAPT) is associated with a well-known genetic form of FTD, and deposits of p-tau are often detected in post-mortem brain of dementia patients [44]. We found extracellular deposits of phosphorylated tau (p-tau, AT8) in the brain of mVCP mice (Supplementary Fig. 1A) which may have formed through non-specific aggregation of proteins. The tau deposits were often observed surrounded by Iba1-positive microglia or GFAP-positive astrocytes, suggesting an inflammatory response to the abnormal presence of p-tau (Supplementary Fig. 1A). In contrast, in control and mVCP mice in which the HSR was amplified by arimoclomol, no tau-positive deposits were observed in any area of the brain assessed.

Whilst TDP-43 plays an important role in RNA metabolism, mislocalisation of this protein suggests that other RNA-associated proteins may also be affected in mVCP mice. Stress granule formation is a highly evolutionarily conserved cytoprotective mechanism to temporarily store stalled translation pre-initiation complexes during episodes of cellular stress [45]. We examined the expression of Tia1, an RNA-binding protein, known to be present in stress granules [46] in mouse brain tissue. We discovered that Tia1 co-localised with both TDP-43 and ubiquitin inclusions in the cytoplasm of mVCP brains suggesting the possible formation of non-specific protein aggregation (Fig. 4C). It has been demonstrated in vitro that TDP-43-containing granules in the cytoplasm may seed aggregation through RNA binding [47, 48]. Furthermore, regenerating myofibres reportedly contain TDP-43 ‘myo-granules’ which may be the precursors for aggregation in diseased muscle, as shown in mutant VCP (A232E) mouse muscle [47]. It is therefore possible that a similar phenomenon also occurs in the brain of mVCP mice, leading to the aggregates observed in this study. Two additional markers of stress granules, FMRP and G3BP, were abnormally observed only in the brain of mVCP mice (Supplementary Fig. 1B). In control animals and mVCP mice treated with arimoclomol, no aggregates positive for any of the tested stress granule markers were observed (Supplementary Fig. 1B).

Furthermore, as observed in the spinal cord, immunostaining of mVCP mouse cortex also revealed the presence of p62 and LC3-positive aggregates, with intense cytosolic LC3 staining (Fig. 5A, B). No LC3 staining was observed in control tissue or tissue from arimoclomol-treated mVCP mice.

Fig. 5

Arimoclomol treatment prevents p62 and LC3 aggregation and enhances HSP70 in mVCP mouse brain. Histological images of (A) p62 expression and, (B) LC3 expression in mouse brain sections. White arrows indicate protein aggregates. (C) HSP70 expression in mouse brain with and without neuronal marker (β-III tubulin, red) and nuclear marker (DAPI, blue). White arrows indicate glial cells expressing HSP70. Scale bar =10μm

Similar to the findings in the spinal cord of mVCP mice, brain tissue from these animals also showed an increase in the expression of HSP70 compared to wtVCP and non-transgenic WT controls, indicative of stress-induced activation of the HSR. In mVCP mice treated with arimoclomol, HSP70 expression was found to be further increased, suggesting that the HSR has been augmented by arimoclomol treatment (Fig. 5C). Interestingly, as noted in the spinal cord, HSP70 expression was significantly pronounced in glial cells in the brain, which are known to have a robust stress induced HSR.

Pathology Observed in mVCP Mice Reflects That Observed in FTD Patient Brain Tissue

To verify whether the pathological features observed in the CNS of mVCP mice, which were improved by amplification of the HSR, have relevance to the human disease, we examined post-mortem cortical brain tissue from 4 patients diagnosed with FTD (see Supplementary Table 1 for further patient information and genetic/clinical sub-classification). A panel of the same markers used in the mVCP mice were assessed and the results compared to samples of the same region from age-matched healthy human brains.

Since autophagy is a common pathway that may be disrupted in FTD brain, we examined human FTD brain for the presence of autophagy-related proteins which were abnormally present in mVCP mice. Globular, juxtanuclear p62-positive cytoplasmic inclusion bodies were present in cortical brain sections of all FTD cases examined (Fig. 6A) and were similar to those seen in mVCP mice (Fig. 5A), suggesting a generalised disruption in protein homeostasis in FTD patients; no p62 immunostaining was observed in brain sections from healthy controls. Strong p62 staining was also observed in the brain of Patient 1 (FTD caused by tau mutation, Fig. 6A), similar to that reported in the brain of Alzheimer’s disease patients early in pathogenesis within neurofibrillary tangles [49]. In Patient 2 (FTD associated with mutant TDP-43), intense p62 staining was observed in neural processes in addition to cytoplasmic inclusion bodies. Interestingly, a similar pattern of TDP-43 staining has been reported in the upper cortical layers in FTD patients with a TDP-43 mutation [50]. Immunostaining of cortical sections also revealed the presence of LC3-positive aggregates in the cortical tissue of all FTD patients (Fig. 6B), similar to findings in mVCP mice (Fig. 5B) which were not seem in control sections.

Fig. 6

FTD patient brain pathology and HSP70 expression levels. (A) p62 immunohistochemistry on post-mortem brain cortex from patients diagnosed with FTD shows p62-positive inclusion bodies. White arrows indicate intensely stained neurites, and black arrows indicate cytoplasmic protein aggregates. DAPI labels nuclei (blue). Scale bar = 10μm unless otherwise indicated. (B) Increased LC3 expression in neurons of post-mortem brain from patients diagnosed with FTD. Darker stained neurons with deposits positive for LC3 observed in all FTD patients. Insets show magnification of marked regions. (C) Cytoplasmic TDP-43 mislocalisation was observed in all FTD patient samples (green), while rarely seen in control tissue. DAPI labels nuclei (blue). Scale bar = 10μm unless otherwise indicated. (D) Western blot of HSP70 expression from FTD patient brain tissue compared to healthy controls and (E) corresponding western blot density bar chart comparing all FTD patient HSP70 levels normalised to controls (t test * p=0.02)

Although TDP-43 mislocalisation in FTD brain is a well-established phenotype, we demonstrate extensive cytoplasmic mislocalisation of TDP-43 in all four FTD patient brains assessed, in which p62 and LC3 are also aggregated. Such mislocalisation was not observed in samples from healthy controls (Fig. 6C). Our findings thus reveal that the pathological phenotypes identified in the mVCP mouse model are indeed present in the human disease. Moreover, these phenotypes are not limited to mutations in VCP, therefore expanding the relevance of our findings of the beneficial effects of targeting the HSR to non-VCP FTD patients.

Whilst the ability to mount a HSR under conditions of cell stress is present throughout life, this cytoprotective mechanism is thought to become less effective in later life, likely contributing to the age-related increase in susceptibility to degenerative diseases [51]. In our study, compared to age-matched control post-mortem samples, HSP70 expression was indeed significantly lower in the cortex of FTD patients (Fig. 6D–E). Therapeutically augmenting this endogenous cytoprotective process may therefore be a beneficial strategy in response to neurodegenerative diseases such as ALS/FTD.

Patient-derived Mutant VCP Fibroblasts and iPSC Motor Neurons Exhibit Pathological Characteristics Which Are Ameliorated by Amplification of the HSR

Our results show for the first time that upregulation of the HSR ameliorates the pathological deficits observed in the brain and spinal cord of mVCP mice. To further test whether the beneficial effects of this approach may have therapeutic relevance for human ALS/FTD, we next examined the effects of treatment with arimoclomol in human models of ALS/FTD by establishing patient-derived cellular models of mutant VCP.

In order to investigate whether patient-derived cells can be used as a platform to test the effects of arimoclomol on VCP-relevant pathology, we initially undertook a preliminary study in mutant VCP patient fibroblasts which have been reported to display a pathological phenotype when grown in culture [52]. In mVCP patient fibroblasts, cultured from four individual patient lines (Fig. 7), cytoplasmic aggregates of TDP-43 were observed (Fig. 7A), and whilst most nuclei stained robustly for TDP-43, some nuclei stained faintly for TDP-43, indicative of nuclear depletion (Fig. 7B). This pattern of staining was observed in all patient lines. No TDP-43-positive cytoplasmic aggregation or nuclear depletion of TDP-43 was observed in control fibroblasts, in which TDP-43 immunostaining was restricted to nuclei (Fig. 7C). In arimoclomol-treated mVCP fibroblasts (10μM, 24-h treatment), the pattern of TDP-43 immunostaining was similar to that observed in healthy controls (Fig. 7D).

Fig. 7

Mutant VCP patient fibroblasts exhibit pathology ameliorated by arimoclomol. Expression of TDP-43 in untreated mVCP fibroblast cultures show (A) TDP-43- positive aggregates or (B) depletion of nuclear TDP-43 (indicated by white arrows). (C) Control, and (D) arimoclomol-treated mVCP cultures show no abnormal TDP-43 expression. Scale bar = 20 μm. (E) DAPI-labelled fluorescent images of abnormal nuclear morphology observed in mVCP patient fibroblasts showing nuclear herniation and nuclear fragmentation generating micronuclei. (F) Quantification of disrupted nuclei in fibroblasts treated with increasing concentrations of arimoclomol, **p<0.01, *** p<0.001 (two-way ANOVA, n=3 controls, n=4 patients)

Furthermore, in untreated mVCP fibroblasts, we also observed a significant increase in the number of nuclei with an abnormal morphology, consisting of herniations and fragmentation of nuclei leading to the generation of micronuclei (Fig. 7E). Surprisingly, these cells were not undergoing apoptosis, as assessed by TUNEL staining for DNA double-strand breaks (Supplementary Fig. 2). Quantification of the number of aberrant nuclei in mVCP fibroblasts in the absence and presence of increasing concentrations of arimoclomol revealed a dose-dependent reduction in the number of disrupted nuclei, with a statistically significant difference observed at 50μM of arimoclomol (Fig. 7F).

Since mVCP fibroblasts successfully demonstrated the rescue of pathological features in human cells by upregulation of the HSR, we began our study in human induced pluripotent stem cell (iPSC)-derived motor neurons (iPSC-MNs) established from ALS patients expressing mutant VCP, which provide a more complex, neuronal and highly disease-specific cell culture model of neurodegeneration, and which have been previously shown to manifest a TDP-43 pathology [53].

In mVCP patient iPSC-derived motor neurons (3 individual patient lines), we observed distinct cytoplasmic TDP-43 staining with many cells also exhibiting nuclear loss of TDP-43 (Fig. 8A, magnified image of cell in mVCP image). Importantly, mislocalised TDP-43 was rarely seen in the mVCP cultures treated with 50 μM arimoclomol and was absent from healthy control cells.

Fig. 8

Human mVCP iPSC-derived motor neurons exhibit pathology ameliorated by arimoclomol. (A) TDP-43 immunoreactivity shows localisation in control, mVCP and arimoclomol-treated mVCP iPSC motor neuron cultures. Arrows indicate cells with cytoplasmic TDP-43. Magnified image of a cell in the mVCP culture with depletion of TDP-43 visible in the nuclear region (inset, arrow indicates depleted region). (B) Fluorescent images of ubiquitin immunoreactivity in mVCP iPSC motor neuron cultures with and without arimoclomol treatment. Arrow indicates ubiquitin-positive aggregate. (C) Bar chart showing percentage of cells with ubiquitin-positive aggregates **p=0.003, **p=0.0001 (one-way ANOVA, n=3 cultures). (D) p62 immunoreactivity in iPSC motor neurons from arimoclomol-treated and untreated mVCP cultures. Arrow indicates p62-positive cytoplasmic protein aggregate. (E) Immunofluorescent images of HSP70 expression with neuronal marker β-III tubulin. DAPI labels nuclei (blue). Scale bar = 10μm.

Ubiquitin-positive and p62 immuno-reactive protein aggregates were also detected in mVCP iPSC-motor neurons, either as many small bodies dispersed throughout the cell or as one large distinct globular aggregate within the cytoplasm (Fig. 8B, D). Quantification showed that there was a significant increase in the number of cells with ubiquitin-positive aggregates in mVCP neurons, from 1.5% in controls to 23.6% in mVCP cultures. In contrast, in mVCP cultures treated with arimoclomol, only 7.5% of neurons contained ubiquitin-positive aggregates (Fig. 8C). These findings corroborate our observations in mVCP mice, where abnormal ubiquitin and p62 accumulation, possibly linked to impairment of autophagy or proteasomal degradation, was present in both the spinal cord and brain. More importantly, these data clearly show that amplification of the HSR leads to a reduction in cytoplasmic ubiquitin aggregates in a specific neurological cellular system with VCP mutation.

As observed in the mVCP mice, HSP70 expression was increased in mVCP iPSC-derived motor neuron cultures under basal conditions, indicating that these cells are under stress and have activated the HSR. Treatment of mVCP iPSC-derived motor neurons with arimoclomol resulted in a clear increase in HSP70 expression above that observed in untreated mVCP iPSC-MNs (Fig. 8E), indicating an enhancement of the endogenous cytoprotective HSR, which is likely to account for the improvement in protein mishandling pathology observed in untreated cells.