Titin is a nucleolar protein in neurons

Titin is the largest protein produced by living cells and its function as a molecular spring in striated muscle is well characterized (1, 2). Here we demonstrate that titin isoforms in the same size range as found in muscle are prominent neuronal proteins in both the central and peripheral nervous systems, including motor neurons in the spinal cord and brain. Within these neurons, titin localizes to the dense fibrillar component of the nucleolus, the site of ribosomal RNA biogenesis and modification, and a critical site of dysfunction in neurodegenerative disease (3–5). Additionally, we show that the levels of both titin mRNA and protein are altered in the spinal cord of SOD1G93A mice, a commonly used model of amyotrophic lateral sclerosis, indicating that titin mediated nucleolar events may in fact contribute to the pathobiology of disease.


Main Text
With a contour length greater than a micron, titin is nearly twice the size of any other polypeptide known (1).Accordingly, the transcript encoding human titin, TTN, is also colossal, comprising over 300,000 nucleotides, and predicted to take more than an hour to transcribe (6).TTN's 363 exons have a coding potential of up to 38,138 residues, or a protein 4200 kDa in size.Although there are potentially thousands of different titin isoforms, only a handful have been functionally characterized.The 3700 kDa N2A isoform and slightly smaller N2B cardiac isoforms are abundant in vertebrate striated and cardiac muscle, respectively, where they provide passive elasticity within the sarcomere (7).
After detecting titin in a neuronal proteomics study (8), we investigated the possibility that titin may in fact function as a bona fide neuronal protein.Here we demonstrate that titin is indeed present in numerous neuronal subtypes including alpha motor neurons, and that it localizes to the dense fibrillar component (DFC) of the nucleolus, the site of ribosomal RNA (rRNA) biogenesis and processing.Given the contributions of nucleolar stress to amyotrophic lateral sclerosis (ALS) (9)(10)(11)(12), and that mutations in the gene encoding titin (TTN) are associated with a rapid functional decline in ALS (13), we posit that titin may be a previously unrecognized piece in the complex puzzle of neurodegenerative disease.

Titin is present in the embryonic and adult nervous system
We detected titin in a previous proteome analysis of embryonic dorsal root ganglia (DRG) (8).To further investigate the possibility that titin may function in the nervous system, we first queried the available literature and various gene and protein expression databases.Surprisingly, both TTN transcript and protein have been detected in multiple neuronal tissues including mouse brain and spinal cord, human brain, and human DRG (8,(14)(15)(16)(17).Next, polyacrylamide gel electrophoresis (PAGE) was performed using both embryonic and adult mouse nervous system tissues and the titin N2A band from rabbit soleus muscle as a size standard (18).Bands in the 3000-3700 kDa range were present in all samples (Fig. 1A), a size unique to titin isoforms.RT-qPCR was next performed to further investigate murine Ttn expression in neurons, and to provide a more quantitative assessment of its expression compared to muscle.As shown in Figure 1B, Ttn transcript is approximately 5-fold more abundant in the embryonic ventricle compared to the DRG, and approximately 700-fold more abundant in adult gastrocnemius muscle compared to the adult pons.Adult spinal cord showed the lowest levels of Ttn message, mirroring its protein levels in the adult spinal cord (Fig. 1A,B).Figure 1.Titin expression in the embryonic and adult nervous system.(A) PAGE) and silver staining of spinal cord (SC) and brain lysates from embryonic and adult mice reveals bands in a the 3000 -3700 kDa range.Titin isoform N2A from rabbit soleus (RS) (3600 kDa) is included as a size standard (18).Bands in the 2200-2300 kDa range in the RS lane are titin degradation products (19).(B) RT-qPCR shows Ttn expression levels in embryonic and adult nervous system tissues relative to muscle.EB, embryonic brain; ESC, embryonic spinal cord; AB, adult brain; ASC, adult spinal cord; V, ventricle; B, brain; NTC, no template control; G, gastrocnemius muscle; C, cerebral cortex; CB, cerebellum; P, pons; M, medulla.

Titin is present in the neuron nucleus
Fluorescent immunohistochemistry (IHC) was next performed to investigate the nervous system cell types that might express titin and to analyze its localization within the cell.Anti-titin antibody 27867-1-AP (Proteintech) shows robust immunoreactivity in tissue sections of mouse muscle.Figure 2A highlights titin's spiral architecture in the sarcomere, confirming 27867-1-AP recognizes native titin.
Since embryonic DRG were used for our previous proteome investigations, tissue sections containing DRG from embryos of the same gestational age (E17.5) were examined first.Robust, punctate spots were visible in the nucleus of neurons within the DRG and the spinal cord ventral horn, with larger neurons exhibiting larger puncti (Fig. 2B-D).Anti-titin antibody 27867-1-AP was raised against the human titin Ig-like domain 149 and likely recognizes Ig-like domain 142 in mouse.This region of mouse titin shares 80% identity (186/232 residues) with the fusion peptide used to raise the antibody (Proteintech, Ag27496).To further test the specificity of the antibody, a pre-adsorption step was performed using titin peptide Ag27496, followed by IHC on tissue sections of muscle, spinal cord and DRG.This pre-adsorption step completely blocked the spiral labeling of muscle and the punctate staining in neuronal cells (Fig. E1A-D), again indicating that 27867-1-AP binds titin.Finally, no fluorescence was observed when IHC was performed in the absence of primary antibody (Fig. E1E-H).
To explore the possibility that in addition to titin, antibody 27867-1-AP may be recognizing a similar, but non-titin epitope, a blast search against all known Mus musculus proteins was performed using the Ag27496 peptide sequence.The top two hits landed on titin isoforms N2-B and N2-A, showing 80% amino acid identity and 99% coverage.The next 15 highest ranking hits were also titin isoforms with percent identities ranging from 68 to 80%.The highest scoring non-titin protein (37% identity, 49% coverage) was obscurin, a non-nuclear protein.These combined data strongly indicate that the nuclear protein recognized via IHC by antibody 27867-1-AP is titin.
As a second approach for investigating titin's localization within the cell, we isolated nuclei from the same nervous system tissue sources as shown in Figure 1, as well as from human spinal cord and DRG (Fig. 2E) and used PAGE to again probe for titin's presence.In agreement with our IHC results showing nuclear localization, bands in the titin size range were detected in these nuclear extracts (Fig. 2F).Titin's size makes its transfer to a membrane particularly challenging.Thus, to confirm their identity, bands corresponding to adult mouse brain and rabbit soleus were excised from the gel and analyzed via LC-MS.
As shown in Table E1, these extremely large bands contain titin.Additionally, increased run times resolved three bands in the 2000-3600 kDa range in both brain and spinal cord lysates including human spinal cord (Fig. 2G,H).

Titin localizes to the nucleolar dense fibrillar component
The punctate pattern of titin immunostaining within a confined region of the neuron nucleus suggested that titin may localize to a specific subnuclear compartment.To further explore this possibility, IHC for titin was combined with 4′,6-diamidino-2-phenylindole (DAPI), a nuclear stain that binds AT rich regions of DNA.Here, titin appeared juxtaposed between bright DAPI+ spots within the fainter DAPI+ nucleus (Fig. 3A-D).Intense DAPI+ puncta mark chromocenters that form perinucleolar heterochromatin and contain nucleolar organizer regions (NORs) that localize around the perimeter of the developing nucleolus (Fig. 3E) (20).The juxtaposition of titin between NORs raised the possibility that titin may localize to the neuron nucleolus.IHC for titin and a marker of the DFC, fibrillarin (FBL) was next employed.Although the pattern of titin and FBL immunofluorescence appeared virtually identical using standard epifluorescence microscopy, laser scanning confocal microscopy (LSCM) demonstrates that the two antibodies recognize partially distinct domains (arrows and arrowheads in Fig. 3F-H   nucleolar localization sequences in human titin (Fig. E2) (24).

Titin is present in multiple neuronal cell types and Schwann cells in the dorsal root ganglia, sympathetic ganglia, and spinal cord
Within the peripheral nervous system, titin is present in the nucleolus of both neurons and Schwann cells (Fig. E3).However, the amount of titin in Schwann cells is dramatically less than in their neuronal counterparts (Fig. E3A-I).IHC using subtype specific antibodies shows that both small-diameter and large-diameter sensory neurons are titin+ (Fig. E3J,K).Robust immunostaining is also present in nucleoli of neurons in the autonomic sympathetic ganglia (Fig. E3L).Within the central nervous system, IHC was performed on spinal cord sections from ChAT-EGFP mice in which EGFP is selectively expressed in cholinergic neurons (Fig. E3M) (25).Nucleolar titin immunostaining was observed in all cholinergic neurons, with large alpha motor neurons in the ventral horn exhibiting the most robust fluorescence and the largest puncti at both embryonic and adult time points (Fig. 2B, E3N-Q).Titin was also observed in smaller GFP+ gamma motor neurons (arrowheads in Fig. E3P,Q).Notably, titin levels are significantly more robust in the cholinergic, EGFP+ subpopulation within the spinal cord compared to EGFP-negative cells with more diffuse DAPI staining (likely interneurons) (asterisks in Fig. E3Q-S) and EGFP-negative glia (cells with triangular or oblong nuclei that stain densely with DAPI (26, 27)) (circled in Fig. SEQ-S) where titin levels are minimal or absent.These findings align with the relative expression of Ttn observed via RT-qPCR; while titin protein is present in most sensory neuron subtypes in the DRG and in Schwann cells, corresponding to its relatively abundant transcript, titin expression is more restricted in the spinal cord, with cholinergic motor neurons exhibiting the highest levels.Since we did not isolate motor neurons from the other cell types present in the cord, it follows that its expression level within the spinal cord as a whole would be quite low.

Titin is present in multiple neuronal cell types in the brain
Within the adult cortex, titin is observed in the nucleolus of M1 and M2 motor neurons (arrows in Fig. E4A-C), but not observed in non-cholinergic, EGFP-negative cells (arrowheads in Fig. E4B).Neurons of the oculomotor, trochlear, trigeminal, and hypoglossal nuclei all exhibit nucleolar titin (Fig. E4D-G).
Finally, titin expression is widespread in neurons of the cerebellum with its amount corresponding to cell size; large Purkinje neurons exhibit the highest titin levels per cell, neurons in the molecular layer exhibit intermediate levels, and the small, tightly packed neurons of the granular layer show minimal amounts of titin (Fig. E4H-L).

Titin may participate in LLPS
Nucleolar scaffolding proteins, including nucleophosmin, undergo heterotypic interactions with other nucleolar components, including proteins that harbor R-motifs and intrinsically disordered regions, to drive liquid-liquid phase separation (LLPS) and formation of the liquid-like condensate of the nucleolus (28,29).LLPS may also direct pre-ribosomal particle assembly within the nucleolus and exit from the nucleolus as part of the ribosome biogenesis process (28).Titin houses hundreds of intrinsically disordered regions as well as numerous R-motifs within its PEVK region, suggesting that titin may participate in LLPS (30).To explore this possibility, we evaluated the PEVK sequence of human titin using six web-based prediction algorithms of phase separation potential (catGRANULE, FuzDrop, ParSe, PScore, PSPer, and PSPredictor).Four algorithms predicted that the PEVK sequence could exhibit liquidliquid phase separation (Table 1).The positive predictions spanned the entire PEVK region, with no clear sub-region identified as being enriched in phase-separation potential (Fig. E5).

Titin levels are altered in SOD1 G93A model mice
Diminished levels of titin protein in the brains of 12-week SOD1 G93A mice were previously reported via mass spectrometry (31).Similarly, we find slightly reduced levels of titin in the cortex of SOD1 G93A mice at the end stage of disease (P120) (Fig. E6).Since lower motor neurons are the most damaged in SOD1 G93A mice (32), we also examined both transcript and protein titin levels in spinal cords of SOD1 G93A mice and nontransgenic (NTG) controls.As shown in Figure 4, although mRNA levels of Ttn are reduced in the mutant by approximately 8-fold, Ttn protein levels are elevated (Fig. 4B, Fig. E7).Using IHC and LSCM we also observed titin's nucleolar pattern in the remaining alpha motor neurons present in SOD1 G93A spinal cords.As shown in Figure 4, the majority of these neurons exhibit a normal nucleolar titin pattern with an intensity comparable to their NTG counterparts (Fig. 4C,D).In addition, although the area of the DFC is more variable in the spinal cords of SOD1 G93A mice, the average area is the same as NTG controls (Fig. 4E).

Discussion
Here we demonstrate that titin resides in the DFC of the neuron nucleolus.Although titin has been detected in previous neuronal proteome and transcriptome studies, its robust presence in the nucleolus of numerous neuronal subtypes is a surprising finding.At 3600 kDa, titin's size may have interfered with its detection previously.
Within the sarcomere, titin adopts a spring-like structure.Although our PAGE analyses of neuronal titin indicate that at least one isoform represents a molecule of similar size, neuronal titin does not appear to adopt the same three-dimensional conformation.Titin's size alone, as well as our analyses demonstrating that it occupies much of the DFC area and spatially overlaps with that of fibrillarin, suggest that titin may serve as a scaffold for formation of the DFC and localization of rRNA modification enzymes.Our computational analyses indicating that titin's intrinsically disordered region has the potential to phase separate are also consistent with a scaffolding function, although additional studies that directly investigate LLPS are needed.Titin's localization to the DFC suggests that in general, titin may contribute to ribosome production and translational homeostasis, two overlapping processes with strong ties to neurodegenerative disease.Intriguingly, polymorphisms in the TTN gene and reduced TTN expression are in fact associated with ALS and an increased severity of disease (13).Although this increase in severity may be due to decreased skeletal muscle function (13), it is also possible that attenuation of titin's function in the neuron nucleolus is the true pathophysiologic driver.Indeed, studies demonstrating that nucleolar stress is an early feature of both familial and sporadic forms of ALS indicate that nucleolar dysfunction may in fact represent a common pathogenic nexus of disease (12).
The majority of ALS cases (~90%) are sporadic, with no family history (33,34).The remaining 10% are inherited and associated with a list of more than 40 genes.Mutations in just four of these genes account for ~70% of familial cases (C9orf72, SOD1, TARDBP and FUS) (35,36).All ALS cases are characterized by insoluble cytosolic protein aggregates in neurons (37,38).Although aggregated TDP-43, the protein encoded by TARDBP, is not present in SOD1-or FUS-related disease, it is a major component of disease aggregates in nearly all sporadic ALS cases (97%) and in most SOD-1-negative familial cases (39,40).
Interestingly, studies have shown that TTN RNA is present in TDP-43 amyloid aggregates that form in muscle during regeneration.These aggregates are structurally similar to the aggregates found in neurons of patients with ALS (41).Given the sheer size of titin's transcript, as well as a plethora of TDP-43 binding sites (41), it is tempting to speculate that titin's transcript might play a role in nucleating TDP-43 and in triggering the formation of aggregates (42).Although the inclusions in SOD1-related ALS are TDP-43negative, it is possible that titin's message may still contribute to the formation of neuronal aggregates that are known to be rich in RNAs.The potential sequestering of Ttn mRNA in aggregates could also explain why Ttn transcript levels are reduced in SOD1 G93A mice.
Containing more than 200 immunoglobulin domains, motifs prone to aggregation (43), titin protein may also contribute to aggregates in ALS.In vitro studies demonstrating the aggregation of specific titin immunoglobulins lend support to this hypothesis (44).Here we show that SOD1 G93A mice have elevated levels of titin protein in the spinal cord.Although cytoplasmic titin aggregates were not detected in motor neurons of SOD1 G93A mice via IHC, it is possible that antibody 27867-1-AP does not recognize misfolded and/or aggregated titin.This could explain how titin protein levels are elevated in SOD1 G93A mice in the context of diminished messenger RNA.Elevated titin levels could also result from a dysfunctional ubiquitin proteasome system in SOD1 G93A mice (45).Our data demonstrating that titin protein levels are not depleted in the spinal cords of SOD1 G93A mice, and that titin patterning appears normal suggests that titin's potential contribution to ALS results from a role other than a diminishment of function.
Here we demonstrate that titin is broadly expressed in neurons of both the peripheral and central nervous systems.Although ALS primarily impacts central nervous system components, multiple studies have shown that peripheral sensory and autonomic neurons are affected as well (46)(47)(48).Thus, our demonstration of titin in peripheral DRG and sympathetic neurons, in addition to central components, is still consistent with a potential role for titin in ALS.Indeed, understanding why alpha motor neurons are most vulnerable to ALS-associated mutations while other neuronal subtypes are less impacted, remains a significant challenge in the field.One obvious difference is size; alpha motor neurons are the largest neurons in the spinal cord.Studies have shown that the number of DFC/FC modules present in the nucleolus is cell type dependent (reviewed in Lafontaine et al.) (21).Given the large size of alpha motor neurons, it follows that they likely have a uniquely large ribosomal burden and correspondingly large number of DFC/FC modules.
Hence, the large difference in the amount of titin observed in neurons compared to other cell types including Schwann cells may simply reflect the much larger ribosomal burden carried by neurons.This ribosomal load may make neurons, particularly large neurons like alpha motor neurons, particularly sensitive to cellular insults that compromise ribosome production.
The unveiling of titin, a colossal protein encoded by an equally colossal transcript, as a nucleolar resident stands to transform our understanding of the molecular functioning and formation of this organelle.
Moreover, given the emerging central role that nucleolar dysfunction plays in neurological disease, a more comprehensive understanding of nucleolar dynamics, including titin's contribution, will almost certainly reveal new avenues for the development of neuroprotective therapies.

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and spinal cord (SC) neurons.(D) shows an 8 enlargement of the boxed region in (C).(E) 9 Nuclei isolated from an adult mouse spinal 10 cord used for PAGE analysis.(F) PAGE 11 analysis of nuclear extracts from mouse and 12 human nervous system tissues generates bands 13 in the 3000-3700 kDa size range.Titin isoform N2A (3600 kDa) from rabbit soleus (RS) (first lane) is shown as a size standard.(G,H) With longer run times, three bands are visible in lysates from adult brain and human SC.EB, embryonic brain; ESC, embryonic spinal cord; AB, adult brain; ASC, adult spinal cord.TUJ1, pan neuronal marker (Beta-III tubulin); DAPI, nuclear marker.Bar = 7.5 um in (A), 45 um in (B), 15 um in (C), 6 um in (D), 35 um in E.
and Movie 1).Overall, LSCM for titin reveals a spherical reticulate pattern that is closely intertwined with FBL and demarcated by DAPI+ NORs.(Fig 3. F-L, Movies 1 and 2).Within the DFC, FBL and other rRNA processing factors cluster into ring-like structures around the fibrillar center (FC)(21,22).These DFC/FC modules are thought to serve as reaction chambers for individual ribosome maturation(21,23).

Figure
Figure3L(asterisk) and Movie 2 show that within the DFC, titin localizes to these clustered rings.Also

Figure 4 .
Figure 4. Titin levels are altered in SOD1 G93A mice.(A) RT-qPCR using three different sets of primers shows that Ttn mRNA levels are diminished by approximately eight-fold in spinal cords of SOD1 G93A