TTLL1 and TTLL4 polyglutamylases are required for the neurodegenerative phenotypes in pcd mice.

Polyglutamylation is a dynamic posttranslational modification where glutamate residues are added to substrate proteins by 8 tubulin tyrosine ligase-like (TTLL) family members (writers) and removed by the 6 member Nna1/CCP family of carboxypeptidases (erasers). Genetic disruption of polyglutamylation leading to hyperglutamylation causes neurodegenerative phenotypes in humans and animal models; the best characterized being the Purkinje cell degeneration (pcd) mouse, a mutant of the gene encoding Nna1/CCP1, the prototypic eraser. Emphasizing the functional importance of the balance between glutamate addition and elimination, loss of TTLL1 prevents Purkinje cell degeneration in pcd. However, whether Ttll1 loss protects other vulnerable neurons in pcd, or if elimination of other TTLLs provides protection is largely unknown. Here using a mouse genetic rescue strategy, we characterized the contribution of Ttll1, 4, 5, 7, or 11 to the degenerative phenotypes in cerebellum, olfactory bulb and retinae of pcd mutants. Ttll1 deficiency attenuates Purkinje cell loss and function and reduces olfactory bulb mitral cell death and retinal photoreceptor degeneration. Moreover, degeneration of photoreceptors in pcd is preceded by impaired rhodopsin trafficking to the rod outer segment and likely represents the causal defect leading to degeneration as this too is rescued by elimination of TTLL1. Although TTLLs have similar catalytic properties on model substrates and several are highly expressed in Purkinje cells (e.g. TTLL5 and 7), besides TTLL1 only TTLL4 deficiency attenuated degeneration of Purkinje and mitral cells in pcd. Additionally, TTLL4 loss partially rescued photoreceptor degeneration and impaired rhodopsin trafficking. Despite their common properties, the polyglutamylation profile changes promoted by TTLL1 and TTLL4 deficiencies in pcd mice are very different. We also report that loss of anabolic TTLL5 synergizes with loss of catabolic Nna1/CCP1 to promote photoreceptor degeneration. Finally, male infertility in pcd is not rescued by loss of any Ttll. These data provide insight into the complexity of polyglutamate homeostasis and function in vivo and potential routes to ameliorate disorders caused by disrupted polyglutamylation.

Introduction

Protein polyglutamylation and de-glutamylation, which are catalyzed, respectively, by tubulin tyrosine ligase-like (TTLL) and cytosolic carboxypeptidase (CCP) enzymes, contribute to diverse biological processes and their mutation underlie rare neurodegenerative disorders. Polyglutamylation consists of the successive enzymatic addition of glutamate molecules to an acceptor glutamic acid within a substrate protein. During polyglutamate chain initiation, the first glutamate is added to the gamma-carboxyl group of a glutamic acid residue. Subsequently, polymer elongation occurs through glutamate addition to the alpha-carboxyl group of this initiating glutamic acid, and continues through iterative alpha linked additions [1]. This is a dynamic process where glutamate polymerization is catalyzed by several TTLL family members, whereas degradation of the polymer is catalyzed by the 6-member CCP family [1-3]. A range of proteins undergo polyglutamylation [4], with tubulin being the first identified and most extensively studied [1,5,6]. Indeed, the substrate specificities of TTLLs and CCPs were mainly determined with tubulin as substrate [1-3,7,8]. TTLL4 and TTLL5 catalyze the formation of initiating γ-carboxyl linkages with preferences for β- and α-tubulin, respectively. TTLL1 and 11 catalyze the formation of α-carboxyl chain-elongating linkages primarily in α-tubulin. TTLL7 catalyzes both initiation and elongation of the polyglutamate side chain of β-tubulin [1,9]. CCP5 uniquely catalyzes removal of the gamma-carboxyl linked branch point glutamate of tubulins [2,10]. The other 5 CCP family members all cleave α-carboxyl linked glutamate residues on tubulin [2,3] although they can be distinguished to some extent by their kinetic properties and synthetic substrate preferences in vitro [10,11] and their non-redundant function in vivo [11-14].

Despite its discovery 30 years ago, only relatively recently has a link been established between disrupted polyglutamylation and neurodegeneration in vivo. This emanated from the recognition that the zinc metallocarboxypeptidase, Nna1 (a.k.a. CCP1) [15], which is mutated in the recessive Purkinje cell degeneration (pcd) mouse [16] is a deglutamylase capable of degrading polyglutamate side chains [2,7,8]. In addition to male infertility, pcd mice are characterized by progressive and selective neurodegeneration of cerebellar Purkinje cells, olfactory bulb mitral cells, thalamic neurons, spinal motor neurons and retinal photoreceptors [17,18]. In parallel, tubulin polyglutamylation levels are increased in the central nervous system and testis of pcd mice [2,7,10]. Mutations of Agtpbp1, the gene encoding Nna1/CCP1, have also been shown to cause lower motor neuron degeneration in sheep [19] and in humans cause an infantile-onset, progressive, and severe neurodegeneration [18,20,21].

A picture is emerging of complex relationships amongst the anabolic and catabolic enzymes involved in polyglutamylation, their substrates and neurodegenerative phenotypes. Elevated tubulin glutamylation, as observed in pcd mice, is associated with neurodegeneration. However, the converse is not true as loss of TTLL1 function results in reduced glutamylation but no neurodegeneration [6] and targeted over-expression of Nna1, CCP4 or CCP6 in Purkinje cells, which degrades polyglutamate chains does not result in their degeneration [11]. These data imply it is longer, rather than shorter, polyglutamate chains that are deleterious to neurons. The role of the balance between anabolism and catabolism of glutamate chains in neurodegeneration is exemplified by the finding that loss of TTLL1 function, which reduces polyglutamylation, rescues Purkinje cell degeneration in pcd mice [7,22].

Many TTLLs are expressed in brain, some at levels similar to, or higher than TTLL1 [5,23,24]. This begs the question of whether additional TTLLs also contribute to neurodegeneration in pcd mice. For example, as TTLL1 mediates elongation of glutamate chains on α-tubulin it might be anticipated that loss of an initiator TTLL for this tubulin isoform such as TTLL5 might produce a similar outcome. Here we test null alleles of different TTLLs for their ability to modify tubulin polyglutamylation and rescue degeneration of retinal photoreceptors, olfactory bulb mitral cells and cerebellar Purkinje neurons as well as testicular structure and function in pcd mice.

Results

Polyglutamate homeostasis and Ttll expression patterns

Glutamylation is a complex balance between glutamate addition by the TTLL family of enzymes and their elimination by the CCP family of carboxypeptidases ([1,2,7,8,14] and Fig 1A). Thus, glutamylation is dependent both on the levels of the various enzymes in particular cells and tissues, their catalytic properties (e.g. initiators or elongators) and preferences for different substrates (e.g. α- vs β-tubulin). The glutamylation homeostatic process is depicted in Fig 1A for α- and β-tubulins. It should be borne in mind that substrate preferences have been largely established in vitro and less is known about their enzymatic properties and specificities in vivo.

Fig 1

Expression and properties of Ttll polyglutamylases.

(A) Schematic representation of anabolic and catabolic enzymes metabolizing polyglutamate chains of tubulin. Initiator and elongator TTLLs differentially prefer α-tubulin (gray) or β-tubulin (brown) [1,5]. CCP5 uniquely cleaves the branching point glutamate, whereas CCP1, 2, 3, 4 and 6 degrade the α-carboxyl linked glutamates in the chain. (B) Expression profiles for Ttlls and Agtpbp1 (gene encoding Nna1/CCP1). Expression was determined using qRT-PCR on total RNA from adult cerebellum (CB), cerebral cortex (CX), olfactory bulb (OB), testis (TS), kidney (KD), and liver (LV). Data represents mean and SEM of transcript copy numbers/ng total RNA from 3 independent mice. Authentic polyglutamylases are indicated in black whereas Ttll3, 8 and 10 (blue) are glycylases [1]and Ttll12 (red) is of unknown activity[1].

To determine which TTLL glutamylases might contribute to the pcd phenotype, we examined their expression by quantitative RT-PCR in cerebellum, olfactory bulb and testis which have abnormalities in pcd mice [25] as well as cerebral cortex, kidney and liver that have no reported deficiencies [17,25] (Fig 1B). In general, expression of Ttlls was highest in testis followed by the three brain regions and lowest levels were observed in kidney and liver. Of note, Agtpbp1, the gene encoding Nna1/CCP1 and is mutated in the pcd mouse, is highly expressed in brain compared to non-neural tissues and is even present at high levels in cerebral cortex which has no reported degeneration.

Loss of Ttll1 or Ttll4, but not Ttll5, 7, or11, attenuates Purkinje cell degeneration in pcd mice

TTLL1, 2, 4, 5, 6, 7, 9, 11 and 13 are proven glutamylases whereas TTLL3, 8 and 10 are glycylases and TTLL12 has no known enzymatic activity [1,26,27]. Of the authenticated glutamylases, expression of Ttll2, 6 and 13 are at the limit of detection in neural tissues and Ttll9 has relatively low expression (Fig 1B). Reviewing the Allen Mouse Brain Atlas and published literature [5,24,28] Ttll1, 4, 5, 7, and 11 are expressed at various levels in cerebellar Purkinje cells and mitral cells of the olfactory bulb that degenerate in pcd mice whereas Ttll9 has marginal or no expression in either neuronal type and no null strain was available to us. Therefore, we focused on Ttll1, 4, 5, 7 and 11 and crossed null alleles of each (S1 Fig) onto a pcd background. Absence of transcripts in all null strains was confirmed by RT-PCR of total RNA from various tissues (Figs 2A, 2B and S2).

Fig 2

TTLL1 and TTLL4 are required for Purkinje cell degeneration in pcd mice.

(A and B) RT-PCR using primers targeting deleted region in Ttll1KO (A) or Ttll4KO (B) allele confirmed the absence of Ttll1or Ttll4 transcripts in cerebellum, brain, eye, and testis of respective KO mice. (C and D) Gender balanced littermates of each genotype (n = 4–10/genotype) at 7 weeks of age were tested on a standardized accelerating rota-rod for five consecutive days. The latency to fall in seconds (mean ± SEM) was not significantly different between WT and Ttll1KO,pcd (C) or Ttll4KO,pcd (D) groups (one-way ANOVA p >0.05), whereas pcd mice were markedly impaired (C and D) (p < 0.05).

All strains of mice were first tested for locomotor performance on an accelerating rota-rod and subsequently sacrificed and survival of Purkinje cells and levels of polyglutamylation in cerebellum determined by immunofluorescence and western blotting, respectively. In pcd mice, only loss of Ttll1 or Ttll4 restored locomotor activity to levels comparable to wild-type mice (Fig 2C and 2D). No significant improvement of rota-rod performance was seen with null alleles of Ttll5, 7 or 11 in pcd mice (S2 Fig).

Degeneration of Purkinje cells in pcd begins around day 17 after birth and progresses over several months and affects some lobes before others [17,25]. Purkinje cell integrity was assessed using immunofluorescence with an anti-calbindin antibody (Fig 3). Low power images reveal that cerebellum of pcd (Fig 3B) is smaller than that of wild-type (Fig 3A) or Ttll1KO,pcd (Fig 3C) or Ttll4KO,pcd mice (Fig 3D). In addition, there is widespread loss of Purkinje cells in 2-month-old pcd mice (Fig 3B and 3B’). In contrast, many calbindin-positive Purkinje cells are evident throughout all cerebellar lobes examined in Ttll1KO,pcd (Fig 3C and 3C’) and Ttll4KO,pcd mice (Fig 3D and 3D’). Higher power images (Fig 3A’–3D’) confirm the presence or absence of Purkinje cells and indicate that the morphology of surviving Purkinje cells does not vary noticeably between wild-type mice and Ttll1KO,pcd or Ttll4KO,pcd mice. The cerebella of Ttll5KO,pcd, Ttll7KO,pcd and Ttll11KO,pcd were also small compared to wild-type mice (S3A–S3D Fig). In addition, deletion of TTLL5, 7 or 11 had no impact on Purkinje cell degeneration in pcd (S3B’–S3D’ Fig).

Fig 3

Loss of TTLL1 and TTLL4 attenuated Purkinje cell degeneration in pcd mice.

(A-D) Calbindin D-28K immunofluorescence staining of cerebellar sections from 2-month old wild-type (A and A’), pcd (B and B’), Ttll1KO,pcd (C and C’) and Ttll4KO,pcd (D and D’). Note the cerebellum of pcd mice (B) is smaller than that of wild-type (A), whereas Ttll1KO or Ttll4KO restored the size of the cerebellum in pcd mice. (A’-D’) 20X view of boxed areas in A-D, respectively showed that calbindin-positive Purkinje neurons are lost in pcd mice (B’), whereas Ttll1KO,pcd (C’) or Ttll4KO,pcd (D’) did not exhibit overt Purkinje cell degeneration. ML: Molecular Layer; PCL: Purkinje Cell Layer; GCL: Granule Cell Layer.

That loss of TTLL1 rescues Purkinje cell death in pcd was known [7,22], but loss of TTLL4 protecting these cells is a novel finding. As Purkinje cell loss is progressive in pcd, we considered the possibility that TTLL4 loss might slow but not prevent their death. Therefore, we compared 10-month-old wild type and Ttll4KO,pcd mice (Fig 4A). The size of cerebellum in Ttll4KO,pcd mice (Fig 4Ab) is not different from wild-type of the same age (Fig 4Aa). Again, many calbindin-positive Purkinje cells were observed in all lobes examined (Fig 4Ab and 4Ad), indicating that the rescue is relatively long term.

Fig 4

Loss of TTLL4 attenuates Purkinje cell degeneration in older pcd mice.

(A) Calbindin D-28K immunofluorescence staining of cerebellar sections from 10-month old wild-type (a and c) and Ttll4KO,pcd (b and d) mice. Note the size of cerebellum of Ttll4KO,pcd is similar to that of wild-type mice and many Purkinje cells survive in Ttll4KO,pcd double mutant (d). (B) Quantitative RNA expression analysis of three Purkinje cell markers, Car8, Pcp2, and Ppp1r17 in cerebella from Ttll1KO (a) or Ttll4KO (b) genotypes. For comparison purposes, data (mean ±SEM) have been normalized to values in wild-type mice Three independent mice per genotype. Expression of the 3 markers is low or undetectable in pcd cerebellum (a and b) reflecting the massive loss of Purkinje cells. In contrast, there are no statistical differences between the expression of the markers in wild-type (WT); Ttll1KO; Ttll4KO; Ttll1KO,pcd and Ttll4KO,pcd mice.

We also wanted to compare the degree of Purkinje cell protection conferred by TTLL1- or TTLL4-deficiency. Anatomical reconstruction of the cerebellum would be a daunting undertaking, so we sought a quantitative orthologous method. Previously, we identified many markers of Purkinje cells using molecular profiling [29]. Therefore, we examined RNA expression of three such markers [30-32] as a surrogate for Purkinje cell numbers in 2 months old mice. Three marker genes were chosen to minimize the possibility that expression of a single marker might be directly affected by glutamylation. Expression of all three markers was almost undetectable in pcd cerebellum (Fig 4B), reflecting the massive loss of Purkinje cells. In contrast expression of the three markers was statistically the same in wild-type (Fig 4Ba and 4Bb), Ttll1KO,pcd (Fig 4Ba) and Ttll4KO,pcd cerebellum (Fig 4Bb). This indicates that the degree of Purkinje cell rescue by loss of Ttll1 in pcd is indistinguishable from that conferred by loss of Ttll4 at this age.

Loss of TTLL1 or TTLL4 variably affect tubulin polyglutamylation in pcd cerebellum

In pcd, tubulin is hyperglutamylated [2] and loss of TTLL1 is reported to reduce glutamylation levels in cerebellum in parallel with its ability to rescue Purkinje cell degeneration [7]. Therefore, we compared tubulin glutamylation levels in cerebellum by immunoblotting of cerebellar extracts using the GT335 (detects gamma-linked branching glutamate [33]) and polyE (detects 3 or more consecutive glutamate residues residing at the C-terminus of a chain [1,6,34]) antibodies. In agreement with previous studies [2,7], tubulin glutamylation is increased in pcd mouse cerebellum and is markedly decreased in Ttll1KO and Ttll1KO,pcd cerebellum (Fig 5A). In contrast, loss of TTLL4 has no marked effect on tubulin polyglutamylation either alone or in pcd mice (Fig 5B). We also quantified the levels of glutamylation and confirm the reduction in Ttll1KO animals and the lack of statistically significant difference between pcd and Ttll4KO,pcd mice (Fig 5A and 5B). Nevertheless, the current analysis does demonstrate that TTLL4 loss of function attenuates Purkinje cell loss up to at least 10-months of age. In addition, we show that although loss of TTLL7 does not spare Purkinje cells in pcd mice, it does reduce polyglutamylation signal (S2C Fig). During revision of this manuscript a study appeared that also demonstrated reduced tubulin polyglutamylation but failure to rescue Purkinje cells in Ttll7KO,pcd mice [35]. These authors also showed that while TTLL1 is considered an α-tubulin preferring enzyme, it can glutamylate β-tubulin in the presence of TTLL7 [35].

Fig 5

Loss of TTLL1 or TTLL4 differentially affects tubulin polyglutamylation in wild-type and pcd cerebellum.

(A) Representative western blot of polyglutamylated tubulins in cerebellar lysates from wild-type (WT), pcd, Ttll1KO, and Ttll1KO,pcd mice detected using GT335 and polyE antibodies. In pcd, there is increased immunoreactivity to both GT335 and polyE antibodies compared to wild-type. However, GT335- and polyE- immunoreactive bands were greatly reduced in Ttll1KO and Ttll1KO,pcd. (B) In contrast to Ttll1KO, loss of Ttll4 function did not alter the basal or elevated polyglutamylation levels in pcd. (C and D) Quantitative analysis of intensity of GT335 or polyE immunoreactive bands using LiCOR with normalization to α-tubulin levels. Whereas Ttll1KO caused a significant reduction in GT335 and polyE signals, loss of Ttll4 had no significant affect. The bars represent the mean ±SEM of 4–6 animals of each genotype. ** p < 0.01; *** p < 0.001; NS: Not significant.

A caveat with immunoblotting is that it is a bulk assay in which Purkinje cells are a relatively minor component and it is possible that there are changes in glutamylation in Purkinje cells that are masked by signal from other cellular components. To address this, we performed immunostaining of cerebellum with the GT335 antibody. As Purkinje cells degenerate in adult pcd, we first examined GT335 staining in postnatal day 19 (P19) mice, prior to Purkinje cell loss. In wild type mice Purkinje cells had prominent GT335 staining, whereas the granule cell layer was only weakly stained (Fig 6A). In pcd and Ttll4KO,pcd mice, there was no obvious difference in the GT335 staining of Purkinje cells compared to wild-type although the granule cell layer was more prominently stained in both (Fig 6B and 6C). To establish specificity of staining we absorbed the antibody with porcine brain tubulin and saw a marked reduction in immunofluorescence (S4 Fig).

Fig 6

Increased GT335 signal in granule cells of pcd mice is not affected by Ttll4 loss of function.

Cerebellar sections from 19 days old (A-C) or adult (D-F) wild-type (A and D), pcd (B and E) or Ttll4KO,pcd (C and F) mice stained with GT335 antibody. (A-C) At P19 prior to Purkinje cell degeneration, GT335-immunoreactivity is prominent in the Purkinje cell soma and principal dendrites of all three genotypes. However, there is no apparent difference in Purkinje cell GT335 immunoreactivity in Ttll4KO,pcd (C) compared with wild-type (A) or pcd (B). In contrast, there appears to be increased GT335 activity in the granule cell layer of pcd and Ttll4KO,pcd mice. (D-F) In adult cerebella, the GT335 immunoreactivity in granule cells remains higher in pcd (E) and Ttll4KO,pcd (F) compared to wild-type (D). As in young animals, there were no obvious differences in GT335 expression pattern in Purkinje cells of wild-type (D) versus Ttll4KO,pcd mice (F). ML: Molecular Layer; PCL: Purkinje Cell Layer; GCL: Granule Cell Layer.

In adult wild-type mice, Purkinje cells have prominent GT335-immunoreactivity particularly in the cell soma and principle dendrites (Fig 6D). In contrast, there are low levels of GT335 immunoreactivity in the granule cell layer. In adult pcd mice, Purkinje cells are absent but the granule cell layer is more intensely stained than in wild-type mice (Fig 6E). In adult Ttll4KO,pcd mice, Purkinje cells are preserved and are GT335-immunoreactive (Fig 6F), although they are not noticeably different from the wild-type. However, the granule cell layer is still intensely GT335-positive (Fig 6F). This suggests the increase in GT355 immunoreactivity on western blots (most likely α-tubulin, based on comparison of its migration with other immunoblotting studies in pcd [2]), represents the contribution from granule neurons. Furthermore, our data indicate that Ttll4 loss does not affect the glutamylation of α-tubulin in granule neurons. In sum, loss of Nna1/CCP1 elicits increased tubulin glutamylation in granule neurons and is independent of TTLL4. However, there are no marked changes in GT335 immunoreactivity in Purkinje cells.

TTLL1 and TTLL4 also contribute to degeneration of olfactory bulb mitral cells in pcd mice

Other neuronal populations degenerate in pcd mice [17,25] and we next determined whether other TTLLs and especially TTLL1 or TTLL4 play any role in this process. Mitral cells of the olfactory bulb degenerate in pcd mice [36] albeit on a slower time course than Purkinje cells [17]. Mitral cells are output neurons central to odorant perception whose dendrites form synapses with olfactory nerve axons in structures called glomeruli and whose axons project via the lateral olfactory tract to the olfactory cortex [37]. We assessed mitral cell survival using Tbr2 immunostaining as a marker (Fig 7) [38]. In 5-month-old wild type mice, Tbr2-positive cells are present in two layers (Fig 7A, 7A’ and 7E). Mitral cells are aligned in a layer (mitral cell layer, MCL) at the junction of the granule cell layer (GCL) and external plexiform layer (EPL) (Fig 7A, 7A’ and arrows in 7E). Tbr2-positive cells (predominantly tufted cells) are also evident in a zone encompassing the boundary of the glomerular (GL) and external plexiform layers (Fig 7A–7D and 7A’–7D’). In pcd mice the olfactory bulb is shrunken and Tbr2-positive cells in the mitral cell layer are markedly reduced (Fig 7B, 7B’ and 7F). In contrast, there is no overt impact on other Tbr2-positive neurons (Fig 7B and 7B’) consistent with earlier studies showing survival of tufted cells in pcd[36]. In both Ttll1KO,pcd (Fig 7C, 7C’ and 7G) and Ttll4KO,pcd (Fig 7D, 7D’ and 7H) mice, there are more Tbr2-positive cells in the MCL compared with pcd mice. There was no overt increased survival of mitral cells in the other TtllKO strains on a pcd background (S5 Fig).

Fig 7

Ttll1 and Ttll4 are required for olfactory bulb mitral cell degeneration in pcd mice.

Sections of olfactory bulbs from 5-month-old wild-type (A-A’, E), pcd (B, B’, and F), Ttll1KO,pcd (C,C’, and G), and Ttll4KO,pcd (D, D’, and H) were immunostained with anti-Tbr2 which recognizes mitral cells and tufted cells. (A-D) low power immunofluorescence images of Tbr2 expression with DAPI counter staining. (A’-D’) Higher magnification images shown in A-D, respectively. Tbr2-positive cells are present in 2 distinct layers. The outer layer is comprised predominantly of tufted cells that are preserved in all genotypes. In contrast the inner layer is predominantly composed of olfactory bulb mitral cells are largely lost in pcd mice at this age (B, B’), whereas concomitant elimination of either Ttll1 (C and C’) or Ttll4 (D and D’) in pcd mice showed more Tbr2-positive mitral cells. (E-H) Bright-field images of IHC also showed that Tbr2-positive mitral cells are lost in pcd mice (compare E to F). Red arrows indicate Tbr2-positive mitral cells in wild type mice and dashed red lines denotes the location of the mitral cell layer in pcd. Note that in Ttll1KO,pcd (G) and Ttll4KO,pcd (H) double mutants more Tbr2-positive mitral cells were evident (red arrows). GL: Glomerular Layer; EPL: External Plexiform Layer; MCL: Mitral Cell Layer; GCL: Granule Cell Layer.

To determine whether loss of TTLL4 function slows rather than prevents mitral cell degeneration, we assessed survival in 7- and 10-month-old wild-type and Ttll4KO,pcd mice. As in younger animals, two bands of Tbr2-positive cells are present throughout the olfactory bulbs of both wild-type and Ttll4KO,pcd mice (Fig 8A and 8B), and many Tbr2-positive cells are present in the MCL of both genotypes at 7 and 10 months (Fig 8). Therefore, as with Purkinje cells in cerebellum, loss of TTLL4 provides a sustained attenuation of olfactory bulb mitral cells loss in pcd mice.

Fig 8

Survival of mitral cells in older Ttll4KO,pcd mice.

Sections of olfactory bulbs from 7-month-old wild-type (A and C), Ttll4KO,pcd (B and D) and, 10-month-old wild-type (E) and Ttll4KO,pcd (F) mice were immunostained with anti-Tbr2 and counter stained with DAPI. Expression of Tbr2 is prominent in two layers throughout olfactory bulb of wild-type (A and C) and Ttll4KO,pcd (B and D) mice at 7-month of age. The same pattern of Tbr2 was evident in olfactory bulb of wild-type (E) and Ttll4KO,pcd (F) at 10-month. GL: Glomerular Layer; EPL: External Plexiform Layer; MCL: Mitral Cell Layer; GCL: Granule Cell Layer.

TTLL1 and TTLL4 differentially contribute to photoreceptor degeneration in pcd mice

We next examined whether knocking out any Ttll attenuated photoreceptor degeneration in pcd mice. Retinae of 5-month-old mice were immunostained for the rod-specific rhodopsin, and nuclei visualized by DAPI. In wild-type mice, the outer nuclear layer (ONL), which comprises the cell bodies of photoreceptors, is 10–12 nuclei deep (~51 μm) and rhodopsin immunoreactivity is restricted to the outer segment layer (OSL) (Fig 9Aa and 9Ae). In pcd, the ONL is much thinner (~ 15 μm) comprising 2–3 nuclei (Fig 9Ab), reflecting substantial photoreceptor loss. In addition, in pcd retinae the OSL is thinner and rhodopsin immunoreactivity is observed not only in the OSL (Fig 9Ab) but also in the ONL (Fig 9Af). The mis-location of rhodopsin is considered a pathological finding indicative of impaired trafficking of proteins from the photoreceptor cell body to the outer segment via the specialized connecting cilium [39,40]. In Ttll1KO,pcd mice, the thickness of the ONL is similar to wild-type values (~10–12 nuclei, ~45 μm), and the length of the OSL is also comparable to that of wild-type (Fig 9Ac) and there is no mis-location of rhodopsin in the cell body (Fig 9Ag). Therefore, at 5-month of age the photoreceptor degeneration in pcd mice is largely prevented by Ttll1 loss-of-function. In the retinae of Ttll4KO,pcd double mutants, the ONL was about 5–7 nuclei in depth (~31μm), which is more than that of pcd but less than wild-type (Fig 9Ad). The OSL in Ttll4KO,pcd retina is also substantially broader than pcd (Fig 9Ad). However, some retention of rhodopsin in cell bodies was evident (Fig 9Ah). Therefore, TTLL4-deficiency partially attenuated or slowed photoreceptor degeneration in pcd mice.

Fig 9

TTLLs contribute to photoreceptor degeneration in pcd mice.

(A) Sections of retinae from 5-month-old wild-type (a), pcd (b), Ttll1KO,pcd (c), and Ttll4KO,pcd (d) mice were immunostained with the rod photoreceptor marker, rhodopsin (green) and nuclei visualized with DAPI (blue). (e-h) Higher power view of rhodopsin staining in the boxed areas in a-d. (a) The outer nuclear layer (ONL) of wild-type retina is about 10–12 nuclei thick, and rhodopsin is exclusively localized in the outer segment layer (OSL) and absent in ONL (e). (b) The ONL of pcd retina is reduced to about 2–3 nuclei in depth and is accompanied by the mis-localization of rhodopsin to cell bodies (f). (c) The number of nuclei in the ONL of Ttll1KO,pcd retina is similar to wild-type and there is no mis-localization of rhodopsin (g). (d) In Ttll4KO,pcd retina, the number of nuclei in the ONL were greater than in pcd (b), but less than wild-type (a) or Ttll1KO,pcd (c) mice. The mis-localization of rhodopsin was still observed in Ttll4KO,pcd retina (h). (B) Sections of retinae from P30 wild-type (a), pcd (b), Ttll1KO,pcd (c), and Ttll4KO,pcd (d) immunostained with rhodopsin (green) and DAPI (blue). (e-h) Higher power image of boxed areas in a-d respectively. In wild-type retina, rhodopsin is exclusively localized in the OSL (a and e). The number of nuclei in the ONL in pcd (b) is comparable to wild-type mice (a), but rhodopsin mis-location to the cell body is already evident (f). In Ttll1KO,pcd retina, there is no rhodopsin mis-location (g). In Ttll4KO,pcd retinae, rhodopsin is mis-located in the photoreceptor cell body (h). (C) Sections of retina from 5-month Ttll5KO,pcd (a), Ttll7KO,pcd (b) and Ttll11KO,pcd (c) were stained as in (A). Loss of function mutants for these Ttlls do not increase the survival of the photoreceptors on the pcd background (compare panel Ab with panels Ca-c). However, in Ttll5KO,pcd retina, there is a near ablation of the ONL (a) with only a single layer of cells containing many gaps. Red and white arrows indicate DAPI positive and rhodopsin positive cells in the ONL. OSL: Outer segment layer; ONL: Outer nuclear layer; INL: Inner nuclear layer.

Rhodopsin retention in the cell body is a sign of aberrant protein transport but may also be the secondary consequence of outer segment degeneration. Therefore, we examined the localization of rhodopsin in retina at age P30, when retina differentiation and maturation are just complete and prior to photoreceptor loss [17,41]. At P30, the depth of the ONL was comparable between the wild-type and pcd retinae (Fig 9Ba and 9Bb). However, rhodopsin was already evident in the cell body of pcd photoreceptors (Fig 9Bf). These results suggest that rhodopsin transport is directly impaired by Nna1 dysfunction in pcd mice. In Ttll1KO,pcd mice no mis-localized rhodopsin was evident (Fig 9Bg). On the other hand, mis-localized rhodopsin was evident in Ttll4KO,pcd retina, although it was less prominent than in pcd mice (Fig 9Bh). Therefore, the aberrant localization of rhodopsin observed in Ttll4KO,pcd mice at 5-month of age is already evident at P30 and confirm eliminating Ttll4 does not fully rescue the deficit in rhodopsin transport in pcd mice.

Besides TTLL1 and TTLL4 loss only TTLL5-deficiency had any influence on photoreceptor degeneration in pcd (Fig 9C). Mutations of TTLL5 have been linked to retinal disease in humans [42] and a Ttll5 mutant mouse is reported to undergo late (~20 months-old) photoreceptor degeneration [43]. At 5-month of age, we did not observe overt loss of photoreceptors in Ttll5KO mice. However, Ttll5KO,pcd mice lose almost all photoreceptors and only a single layer of cell bodies survives in the ONL (Fig 9Ca). Thus, loss of anabolic TTLL5 synergizes with loss of catabolic Nna1 to promote photoreceptor degeneration.

Neither Ttll1KO nor Ttll4KO rescues male infertility in pcd mice

The pcd male is infertile due to defects in spermatogenesis associated with testicular structural anomalies and cell death in the germinal epithelium [10,44]. TTLL1 (the gene targeted here) is the catalytic subunit of a multiprotein complex [26] and a mutation in another component of the complex (PGs1) behaves as a TTLL1-loss of function allele [24,45]. The PGs1mutant also exhibits male infertility and has testicular anomalies [45]. Progeny testing showed that the Ttll1KO males used here were also infertile. Cell death in the germinal epithelium was characterized by the presence of pyknotic and/or multinucleated giant cells, the latter being a specific form of germ cell degeneration caused by the fusion of spermatids (Fig 10, compare panel Aa with panels Ab, Ac, Dd and Ff; arrows indicate dying cells). Given that depletion of Ttll1 or Ttll4 ameliorated neuronal degeneration in pcd mice, we assessed whether knocking out Ttll1 or Ttll4 could also rescue male infertility.

Fig 10

Neither Ttll1 nor Ttll4 knockouts rescued male infertility of pcd mice.

(A) Hematoxylin and eosin staining on sections of testis from 3-month-old wild-type (a and a’), pcd (b and b’), Ttll1KO (c and c’), Ttll1KO,pcd (d and d’), Ttll4KO (e and e’), and Ttll4KO,pcd (f and f’) mice. Compared to wild-type (a and a’) mice, the elongating spermatids were reduced in pcd testis (b and b’). Knocking out Ttll1 led to destruction of the germinal epithelium and cell loss in testis (c and c’), and partially restored testicular morphology in pcd mice (d and d’), although marked cell loss was still evident (arrows). Ttll4KO did not alter the general morphology of testis compared to that of wild-type testis (e and e’), and had a marginal effect in increasing the thickness of the germinal epithelium and the diameter of seminiferous tubules in pcd (f and f’) but cell death was still evident (arrows). (B) Sperm counts from epididymis and vas deferens of wild-type; pcd; Ttll4KO; Ttll4KO,pcd; Ttll1KO and Ttll1KO,pcd mice. Ttll4KO mice are fertile, and their sperm counts are indistinguishable from wildtype (p = 0.19) (Student’s t test). In contrast, Ttll1KO and pcd mice are infertile and both have low sperm counts compared to wild type (p = 0.002, in both cases). Elimination of Ttll1 or Ttll4 failed to increase the sperm counts of pcd mice (Ttll1KO,pcd, p = 0.25; Ttll4KO,pcd, p = 0.20).

Hematoxylin-eosin staining revealed a striking cell loss in the germinal epithelium of Ttll1KO testes (Fig 10 Ac and 10Ac’s, arrows indicate dying cells). These morphologic changes were more severe than those reported for PGs1 mutants [45] and even those of pcd (Fig 10Ab and 10Ab’). The Ttll1KO,pcd testis, although still abnormal, had substantially improved overall morphology and reduced cell loss in the germinal epithelium (Fig 10Ad and 10Ad’) compared to either Ttll1KO or pcd. As expected, the sperm count of Ttll1KO mice was significantly lower than that of the wild-type (Fig 10B). However, despite the improved testicular cytoarchitecture, the sperm count from Ttll1KO,pcd double mutants was not significantly different from that of either Ttll1KO or pcd (Fig 10B).

In contrast to Ttll1 mutants, progeny testing showed that male (and female) Ttll4KO mice were fertile. Indeed, the morphology of the testes and sperm counts from Ttll4KO mice were comparable with that of wild-type littermates (Fig 10Ae, 10Ae’, 10Aa, 10Aa’ and 10B). The testicular structure in Ttll4KO,pcd double mutants (Fig 10Af and 10Af’) was improved compared with pcd mice, but substantial numbers of dead cells were still evident (Fig 10Af’ arrows). Despite the improved testicular morphology, the sperm count from Ttll4KO,pcd was still as low as that of pcd mice (Fig 10B).

Discussion

A growing body of work has demonstrated that appropriately regulated polyglutamylation is required for neuronal survival across species [2,18,19] and also contributes to axonal regeneration [15,46] and spermatogenesis [10,27,45,47]. In this study we investigated the contribution of anabolic TTLL enzymes to polyglutamate homeostasis and degenerative phenotypes in pcd mice that are deficient in the catabolic Nna1/CCP1 enzyme [2,16]. We show that TTLL1 and TTLL4 are unique among the polyglutamylases as their loss counteracts to varying degrees the degenerative phenotypes in cerebellum, olfactory bulb and retinae of pcd mice (this study and [7,22]); providing evidence that the degeneration of different types of neurons in pcd mice likely share a common molecular pathogenesis involving hyperglutamylation.

An important question is the identity of the relevant substrate(s) for TTLL1 and TTLL4. The preference of TTLL1 for α-tubulin and TTLL4 for β-tubulin suggests that hyperglutamylation of both tubulin subunits is required for degeneration in pcd. However, this notion raises several questions. TTLL4 and TTLL7 are both expressed in Purkinje cells [5,28] and both initiate glutamylation of β-tubulin yet loss of TTLL7 does not counteract Purkinje cell degeneration in pcd (this study and [35]). Moreover, loss of TTLL7 reduces tubulin glutamylation in pcd (this study and [35]), whereas TTLL4 loss has no discernible effect either in bulk western blots or immunofluorescence analyses (Figs 5 and 6). This questions whether hyperglutamylation of β-tubulin contributes to the pcd phenotype and indeed whether tubulins are the relevant substrate for TTLL4 in this model. Non-tubulin substrates for polyglutamylation have been identified [4,43,48] but whether any of these contribute to degeneration is unknown. A similar specificity paradox exists for TTLL1 and TTLL5 which are both expressed in Purkinje cells [28]and prefer α-tubulin [1,26], yet only TTLL1 loss prevents degeneration (Fig 3). There are caveats for interpreting these data. First, substrate specificities and catalytic properties of TTLLs should be more accurately viewed as preferences that were established largely in vitro [1,26] and with much less being known about their selectivity in vivo in specific cell types. Indeed, a recent study found that TTLL1, which is generally considered an elongator for α-tubulin, may function as an elongator on beta-tubulin in vivo [22,35]. Second, as proposed in the tubulin code, the various tubulin isoforms may be glutamylated at multiple sites [49,50] which could have distinct functional outcomes depending upon the pattern of residues glutamylated the lengths of the chains and the status of other tubulin post-translational modifications such as glycylation [51]. A further possibility is that critical glutamylation changes are confined to specific regions of the cell. To assess these parameters in vivo with single cell and sub-cellular specificity is challenging with the reagents and technologies currently available. Therefore, the conclusions that hyperglutamylation of β-tubulin is pathogenic and that it is the critical target substrate for TTLL4 in vivo requires further investigation.

If there is a common underlying pathogenic mechanism involving hyperglutamylation several observations remain to be explained. First, Nna1/CCP1 is broadly expressed in CNS neurons [16] and its elimination in pcd results in hyperglutamylation of tubulin throughout brain, yet only specific subsets of neurons degenerate [17]. Second, the tempo of degeneration of different cell classes varies, with some dying early, such as Purkinje cells whereas others, such as olfactory bulb mitral cells degenerate on an intermediate time scale and yet others, such as retinal photoreceptors, die progressively throughout a more protracted timeline [17,25]. How and whether these properties reflect differences in the balance of polyglutamate homeostasis (e.g. levels of various TTLLs and CCPs) or levels and subcellular distributions of different tubulin isoforms or other substrates in various cell classes remains to be fully established. Our systematic genetic analysis of TTLL dependencies in pcd mice provides perspectives on these issues.

Purkinje cells and mitral cells have relatively high levels of both TTLL1 and TTLL4 and both TTLLs contribute to degeneration in pcd. Therefore, intrinsic differences in the expression of TTLL1 and TTLL4 in distinct neuronal populations may be one determinant of their relative susceptibility to damage in response to perturbations that promote hyperglutamylation. Such intrinsic differences in TTLL1 and TTLL4 expression could also underlie the tempo of neuronal degeneration. Experiments are underway to test this hypothesis.

The degeneration of retinal photoreceptors highlights additional characteristics of the involvement of polyglutamylation in this process. Retinal photoreceptors appear particularly sensitive to changes in glutamylation status as their degeneration occurs not only in pcd, but also in mice or humans deficient in CCP5 [52,53] or TTLL5 [42,43] whose losses have no overt effects on other neurons [43]. In this instance sensitivity may relate to the specialized cellular architecture of photoreceptors. The rod outer segment of photoreceptors has no intrinsic translational capacity and proteins necessary for outer segment structure and function must be transported from the inner segment via a specialized connecting cilium [40]. The correct structure and function of the connecting cilium is essential for photoreceptor integrity and mutations in genes encoding its constituent proteins underlie several retinal diseases [39]. Furthermore, polyglutamylated tubulin is known to be associated with the axoneme of the connecting cilium [51] as well as cilia in general, centrioles, basal bodies, and flagella [54] and play a role in the proper formation of ciliated structures and trafficking along the cilia [55-57]. Indeed, hyperglutamylation of tubulin is associated with altered trafficking of vesicles and organelles in neurons of pcd mice although a causal relationship to degeneration has yet to be established [22]. The early-onset mis-localization of rhodopsin to the photoreceptor cell body is regarded as an indicator of impaired trafficking through the connecting cilium [40] and is a prominent feature of the pcd retina both prior to and during receptor degeneration (Fig 9 and [51]). Our finding that TTLL1 deficiency not only attenuated photoreceptor loss but also eliminated the mis-localization of rhodopsin prior to receptor degeneration in pcd indicates it restores normal levels of transport through the connecting cilium and that impaired transport is the underlying cause of the degeneration. This notion is further supported by our finding that TTLL4-deficiency does not provide the same level of attenuation of photoreceptor loss as TTLL1 loss and only partially improves rhodopsin mis-localization (Fig 9). Given the ongoing impairment of rhodopsin trafficking in pcd,Ttll4 KO mice it is probable that its loss of function slows rather than prevents photoreceptor degeneration in pcd mice. It also indicates that if TTLL1 and TTLL4 act in a common pathway the two are not equipotent, at least in the retina.

Mutations of the X-linked retinitis pigmentosa GTPase regulator (RPGR) cause photoreceptor degeneration [58]. Furthermore, a specific isoform of RPGR is a substrate for TTLL5 [43] and mutations in TTLL5 cause retinal dystrophy in humans [42,59] and slow photoreceptor degeneration in mice leading to the proposal that glutamylation of RPGR is required for its normal function [43]. We did not observe any noticeable photoreceptor loss in 5-month-old TTLL5-null mice, which is unsurprising as the reported loss was in mice of ~20-months of age [43]. However, 5-month-old Ttll5KO,pcd double mutants had a more severe photoreceptor loss compared to pcd alone (Fig 9C). Together this highlights two points: first, hyperglutamylation of one substrate can act cooperatively with hypoglutamylation of a different substrate to elicit photoreceptor degeneration; second, the tempo of degeneration is slower for hypoglutamylation compared to hyperglutamylation.

In testis, all enzymes involved in polyglutamylation are highly expressed [5] (Fig 1), potentially reflecting the importance of tubulin polyglutamylation in, for example, sperm flagellum formation. Despite their abundance, only the anabolic TTLL1, TTLL5, and TTLL9 [27,45,47], and the catabolic Nna1/CCP1 and CCP5[3,10,44] play key roles in spermatogenesis. In this study, Ttll1 deficiency caused the most striking defects in testicular structure and morphology, worse even than pcd (Fig 10Ab and 10Ac). The abnormal morphology was accompanied by very low sperm counts (Fig 10B). In Ttll1KO,pcd double mutants, testicular morphology was improved compared to that of either single KO (compare Fig 10Ad with Fig 10Ab and 10Ac), however the marked deficit in sperm numbers was not rescued (Fig 10B). Therefore, TTLL1 and Nna1/CCP1 counteract each other in some but not all aspects of testicular function. In contrast to Ttll1KO mice, Ttll4KO males are fertile with no obvious testicular anomalies and normal sperm counts (Fig 10Ae and 10B). Nevertheless, knocking out Ttll4 also rescued much of the aberrant testicular morphology in pcd (Fig 10Ab) but failed to rescue sperm counts (Fig 10B). This suggests that TTLL4 also generates substrates for Nna1/CCP1, but its function is dispensable in testis. Thus, the products of multiple TTLL polyglutamylases concomitantly contribute to male infertility in pcd mice.

Tubulin polyglutamylation levels in testis are generally low [10]. During male gametogenesis, exquisite regulation of tubulin posttranslational modification is required in multiple events such as spindle cleavage in mitosis and meiosis, sperm flagellum formation, trafficking in germ and Sertoli cells, and manchette formation and dismantlement [60]. It is conceivable that TTLL activity may be required in a transient manner during different events of spermatogenesis. This might be achieved through transcriptional regulation of their expression and potentially allosteric mechanisms as well as by the levels and activity of CCPs that metabolize their substrates. In contrast to markedly impaired male reproductive capacity, female reproduction is essentially normal in all TtllKO strains examined. This is despite the fact that tubulin posttranslational modification plays a role during oocyte maturation [60] and motility of cilia in the Fallopian tube epithelium is required for female reproduction [61].

This study identified the functional relationships between TTLL polyglutamylases and Nna1 in the nervous and male reproductive systems and revealed that only TTLL1 and TTLL4 generate the Nna1 substrates essential for neuronal degeneration. Further characterization of the substrates may provide insight into whether disrupted polyglutamylation plays a broader role in neurodegeneration and male infertility and present therapeutic opportunities.

Materials and methods

Ethics statement

All studies were approved by the St. Jude Children’s Research Hospital (SJCRH) Animal Care and Use Committee (ACUC) and complied with the standards set forth in National Institute of Health Guide for the Care and Use of Laboratory Animals (NIH Publication No. 80–23, revised 1996).

Animals

The pcd (Purkinje cell degeneration 3 Jackson:BALB/cByJ-Agtpbp1/J) mice were purchased from the Jackson Laboratory (Bar Harbor, ME, USA). The Ttll1KO (B6.129-Ttll1/SetoRbrc), Ttll5KO (B6.129-Ttll5/SetoRbrc), Ttll7KO (B6;129-Ttll7/SetoRbrc), and Ttll11KO (C57BL/6N-Ttll11/SetoRbrc) alleles were obtained from Riken BioResource Center (Catalog numbers are RBRC03327, RBRC03328, RBRC03329, and RBRC03337, respectively). A conditional knock-out allele of Ttll4 (B6Dnk;B6N-Ttll4tm1a(EUCOMM)Wtsi/H) was obtained from EMMA (ID: EM:04238), which was crossed with Sox2-Cre (B6.Cg-Edil3Tg(Sox2-cre)1Amc/J) transgenic mice (Jackson Laboratory, Stock Number: 008454) to generate a constitutive Ttll4KO allele. The strategies to generate KO alleles of all Ttll used in the study were summarized in S1 Fig. The Ttll and pcd double knockout mice are referred to as TtllKO,pcd. Animals were maintained on a 12-h light: 12-h dark cycle with free access to food and water.

Generation and genotyping of TtllKO and TtllKO,pcd double mutants

The heterozygotes of each Ttll were bred with pcd heterozygotes to generate Ttll,pcd double heterozygotes, which were further inbred to obtain individual TtllKO,pcd double knock-out mice, wild-type littermates, and other intermediate genotypes. Genotyping of pcd was done as described previously [16,29]. Genotyping the wild-type and TtllKO alleles was performed by PCR using the primers listed in S1 Table. Genotyping was also confirmed at the RNA level using RT-PCR on total RNA extracted from cerebellum, cerebral cortex, testis, or eye. First strand cDNA was generated using Superscript III kit and amplified with primers that target the deleted region of the respective KO alleles (primers listed in S2 Table). β-actin was amplified as a loading control using primers described previously [11].

qRT-PCR analysis of Ttll expression

The levels of Ttll1-13 and Agtpbp1 mRNAs from cerebellum, cerebral cortex, olfactory bulb, testis, kidney and liver were evaluated by qRT-PCR. Total RNA was isolated using Trizol (ThermoFisher) and cDNA synthesized using random hexamers and the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) according to the manufacturer’s instructions. Ttll1-13 and Agtpbp1 mouse-specific TaqMan Gene Expression Assays were purchased from ThermoFisher (Ttll1, Mm00626796_m1; Ttll2, Mm03057809_g1; Ttll3, Mm00505799_m1; Ttll4, Mm01259116_m1; Ttll5, Mm00662072_m1; Ttll6, Mm00555632_m1; Ttll7, Mm01311964_m1; Ttll8, Mm00555808_m1; Ttll9, Mm00512463_m1; Ttll10, Mm01263887_m1; Ttll11, Mm01336822_m1; Ttll2, Mm00812915-m1; Ttl13, Mm00625131-m1; and Agtpbp1, Mm00508131_m1). qRT-PCR was performed using 2 X TaqMan Fast Reagents Starter Kit (Applied Biosystems) according to the manufacturer’s instructions, using the ABI 7900 Fast Real-Time PCR system (Applied Biosystems). PCR conditions were as follows: uracil-N-glycosylase incubation, 50°C for 2 min, AmpliTaq Gold activation, 95°C for 10 min, denaturation step 95°C for 15 s, annealing step 60°C for 1 min; 40 cycles were performed. Standards for absolute quantification were obtained by cloning Ttll1-13 and Agtpbp1 into pcDNA3.1 vector. PCR results were normalized to beta-actin (Actb) [62].

Histology and immunohistochemistry

The procedures for histological analyses of cerebella, retina, olfactory bulbs, and testes were conducted as described [44,63,64]. A rabbit anti-calbindin D-28K antibody (Chemicon, Temecula, CA, USA) and a rabbit anti-Tbr2 antibody (Abcam, Cambridge, MA, USA) were used at the dilution of 1:500 to visualize Purkinje cells and olfactory bulb mitral cells, respectively and immune complexes were revealed using a peroxidase-conjugated anti-rabbit kit and diaminobenzidine tetrahydrochloride (DAB) substrate (Vector Labs, Burlingame, CA, USA). After immunostaining, sections were counterstained with hematoxylin (Sigma-Aldrich, St Louis, MO, USA).

Immunofluorescence

Brain

Sagittal sections from paraffin embedded mouse brain were cut at a thickness of 5 μm. After deparaffinization, matched sections were heat retrieved in 0.01M sodium citrate buffer (pH 6.0) containing 0.05% Tween-20 (Sigma, St. Louis, MO) for 20 min at 98°C. After blocking with 10% normal horse serum (Vector Labs, Burlingame, CA) in PBS, sections were incubated overnight at 4°C with antibodies to Calbindin D-28-K (1:1,000, EMD Millipore, USA), Tbr2 (1:400, Abcam), or GT335 monoclonal antibody (1:1,000, Adipogen, San Diego, CA, USA) or GT335 antibody pre-absorbed with porcine brain tubulin (Cytoskeleton Inc, Denver, CO), then incubated for 1 h at room temperature with Alexa 488-labeled donkey anti-mouse or anti-rabbit antibody (1:200, Invitrogen, San Diego, CA). Sections were counterstained with DAPI (Invitrogen, San Diego, CA).

Retina

For 5-month old mice, eyes were dissected, fixed in 4% paraformaldehyde, and embedded in paraffin after sequential dehydration. Immunofluorescence was performed on 5 μm sections. For postnatal day 30 (P30) mice, the retinae were peeled off from eyes and fixed in 4% paraformaldehyde before embedding in OCT embedding medium (Sakura Finetek, Torrance, CA, USA). Immunofluorescence was performed on 12 μm sections. A mouse anti-rhodopsin antibody [41] was used at the dilution of 1:500 to determine the localization of rhodopsin, which is visualized using Alexa Fluor 488 goat anti-mouse IgG (1:500). Images were taken with a Zeiss LSM 710 NLO Confocal Microscope or an Olympus BX60 equipped with DP71 camera. Sections from retina that included the optic nerve were chosen for comparison.

Protein electrophoresis and immunoblotting

Proteins were separated using a Criterion XT precast gel (4–12% Bis-Tris, (Biorad, Hercules, CA, USA)). After electrophoresis, proteins were transferred onto a nitrocellulose membrane using the Criterion Blotter (Biorad, Hercules, CA, USA). Membranes were incubated with mouse anti-glutamate (GT335, 1:4000, Adipogen), rabbit anti-long-chain polyglutamate (polyE, 1:4,000, Adipogen), or rabbit anti-α-tubulin (EP1332Y, 1:3000, Abcam) antibodies. Immunoreactivity of proteins was visualized with Supersignal West Pico Chemiluminescence Substrate (Thermo, Rockford, IL, USA) following incubation with HRP-labeled sheep anti-mouse (1:2000, GE Healthcare Sciences, Pittsburgh, USA) or donkey anti-rabbit IgG (1:10,000, GE Healthcare Sciences) antisera.

Microarray hybridization and analysis

Cerebella of 2-month-old mice were collected and RNA was isolated according to the manufacturers’ protocol (TRIzol, ThermoFisher). RNA quality was confirmed by analysis on the Agilent 2100 Bioanalyzer. Total RNA was processed in Hartwell Center for Bioinformatics & Biotechnology at St. Jude Children’s Research Hospital according to the Affymetrix GeneChip eukaryote two-cycle target labeling protocol. Biotin-labeled cRNA was added to a hybridization cocktail and then processed automatically on Mouse Genome 2.0_2 or Mouse Clariom S array. Normalized transcript measures were generated from scan intensity files using the RMA algorithm. The array data was quantitated for three Purkinje cell enriched transcripts (Pcp2, Ppp1r17 and Car8) that were identified previously [29] in wild-type; pcd; Ttll1KO; Ttll4KO; Ttll1KO,pcd, and Ttll4KO,pcd cerebella.

Sperm count

Sperm from epididymis and vas deferens were counted using the protocol described previously [65] with minor modifications by including sperm from vas deferens in counting.

Rota-rod test

To assess motor coordination, balance, and motor learning, gender- and age-matched littermate mice were tested on a rota-rod (San Diego Instruments, San Diego, CA) with an accelerating speed (0 to 40 rpm in 4 min and then hold constant speed for an additional min) as described previously [11,63], and the latency of the mice falling from the rod was scored as an index of their motor coordination. When both locomotor coordination and motor learning were assessed, the test was conducted for 5 consecutive days.

Statistical methods

The latencies to fall in the rota-rod test were expressed as mean ± SEM (in seconds) and were analyzed for statistical significance using One-way analysis of variance (ANOVA) with repeated measures followed by Bonferroni’s multiple comparison test or Student’s t-test for comparison between samples at the same time point. The level of significance was set at p < 0.05. In all other experiments, Student’s t test was used to compare independent samples for statistical significance. Significance was set at p of < 0.05. Student’s t test was performed using Microsoft Excel software.

Primers used for genotyping the knock-out alleles of TTLL.

(DOCX)

Click here for additional data file.

Primers used in RT-PCR to determine the transcripts of TTLL.

(DOCX)

Click here for additional data file.

Numerical data for all figures.

(XLSX)

Click here for additional data file.

Schematic representation of knock-out strategy of Ttll members.

The regions encoding the TTL domain in Ttll1 (A), Ttll5 (C), Ttll7 (D), and Ttll11 (E) were replaced with a neomycin (neo) selection cassette. (B) A conditional knock-out allele of Ttll4 where exons 6–8 were flanked with loxp sites was crossed with the maternally expressing Sox2-cre transgenic mice to create Ttll4KO mice with constitutive deletion of exons 6–8.

(TIF)

Click here for additional data file.

Loss of Ttll5, 7, or 11 did not rescue the locomotor deficit in pcd mice.

(A) RT-PCR using primers targeting deleted region in Ttll5, 7 or 11KO allele confirmed the absence of Ttll5, 7, or 11 transcripts in cerebellum, brain, and testis of respective KO mice. (B) Rota-rod test of 2-month-old gender-balanced littermates of wild-type; pcd; Ttll5KO,pcd;, Ttll7KO,pcd;, and Ttll11KO,pcd (n = 4–17/genotype) revealed that loss of function of these genes did not improve the locomotor deficit in pcd mice. (C) Tubulin polyglutamylation levels in cerebellar lysates from wild-type (WT), pcd; Ttll5KO; Ttll5KO,pcd; Ttll7KO; Ttll7KO,pcd; Ttll11KO and Ttll11KO,pcd mice. Loss of function of Ttll5 and Ttll11 had little effect on polyglutamylation whereas, loss of function of Ttll7 in pcd markedly reduced GT335 signal, although it did not improve locomotor score.

(TIF)

Click here for additional data file.

Loss of Ttll5, 7, or 11 did not rescue Purkinje cell loss in pcd mice.

(A-D) Calbindin D-28K immunofluorescence staining of cerebellar sections from 5-month-old wild-type (A and A’), Ttll5KO,pcd (B and B’), Ttll7KO,pcd (C and C’) and Ttll11KO,pcd (D and D’) mice. Note the cerebellum of all TtllKO,pcd mice strains (B-D) is smaller than that of wild-type (A). (A’-D’) Higher magnification of boxed areas in A-D, respectively showed that calbindin-positive Purkinje neurons are not restored in Ttll5KO,pcd, Ttll7KO,pcd or Ttll11KO,pcd mice. ML: Molecular Layer; PCL: Purkinje Cell Layer; GCL: Granule Cell Layer.

(TIF)

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GT335 antibody specificity.

Sections of adult cerebellum were immunostained with GT335 antibody (green) without (A) or with (B) porcine tubulin pre-absorption and nuclei visualized with DAPI (blue). Note large reduction in immunoreactive signal with the pre-absorbed antibody.

(TIF)

Click here for additional data file.

Knocking out Ttll5, 7, or 11 did not rescue olfactory bulb mitral cell degeneration in pcd mice.

Sections of olfactory bulbs from 5-month-old wild-type (A, A’, E), Ttll5KO,pcd (B, B’, and F), Ttll7KO,pcd (C, C’, and G), and Ttll11KO,pcd (D, D’, and H) mice were immunostained for Tbr2, which recognizes mitral cells and tufted cells located in 2 distinct layers. In all genotypes, Tbr2-positive cells are present in the outer layer where tufted cells are located (A’-D’), whereas Tbr2-positive mitral cells are present in wild-type (A’), but almost completely absent in Ttll5KO,pcd (B’), Ttll7KO,pcd (C’), and Ttll11KO,pcd (D’) mice. (E-H) Bright-field immunohistochemistry images showed that Tbr2-positive mitral cells are present in wild-type mice (E), but largely absent in Ttll5KO,pcd (F), Ttll7KO,pcd (G), or Ttll11KO,pcd (H) double mutants. Arrows indicate Tbr2-positive cells in the MCL and dotted red lines indicate position of MCL. GL: Glomerular Layer; EPL: External Plexiform Layer; MCL: Mitral Cell Layer; GCL: Granule Cell Layer.

(TIF)

Click here for additional data file.

28 Apr 2021

Dear Dr Rong,

Thank you very much for submitting your Research Article entitled 'TTLL1 and TTLL4 polyglutamylases are required for the neurodegenerative phenotypes in pcd mice' to PLOS Genetics.

The manuscript was fully evaluated at the editorial level and by independent peer reviewers. The reviewers appreciated the attention to an important problem, but raised some substantial concerns about the current manuscript. Based on the reviews, we will not be able to accept this version of the manuscript, but we would be willing to review a much-revised version. We cannot, of course, promise publication at that time.

Each of these expert reviewers thought your manuscript holds broad interest and potentially significant impact, but as per several concerns it seems that a key take-home message - that TTLL4 but not the other paralogues rescues the CCP1 ko phenotype - was significantly missed or lost, due to a number of factors including data presentation and analysis that falls short, and unclear writing/organization. In addition, as Reviewer 1 noted, the mere fact that TTLL6 and 11 are not expressed highly seems to undermine the observation of non-rescue by these genes. As a result, you will need to make significant revisions - satisfying the reviewers with a combination of revised writing/emphasis, new data or new data analysis - before the manuscript can be considered further. While some of the concerns are more important than others, we will require a detailed list of your responses to the review comments and a description of the changes you have made in the manuscript as we will put any revised manuscript back in their hands.

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Reviewer #1: The levels of polyglutamylation have been shown to be critical for neuronal survival and function. Loss of CCP1 (as in pcd mice), an enzyme that removes this modification, results in the degeneration of select neuronal populations in mice, indicating that hyperglutamylation is particularly problematic. Previous studies have shown that loss of TTLL1, a tubulin tyrosine ligase-like enzyme, which adds glutamate residues to substrate proteins can protect Purkinje cells in pcd mice. Here, Wu et al. expand on these findings, investigating the contribution of TTLL1 to degeneration of other neuronal populations in pcd mice. They also examine the contribution of 5 other members of the tubulin tyrosine family to phenotypes in pcd mice. This systematic genetic analysis can shed light on the contribution of these different enzymes to cellular homeostasis and survival and is thus of interest to the readers of Plos Genetics. However, there are a number of concerns that must be addressed before the paper is suitable for publication.

Major concerns:

1. The authors show that loss of TTLL6 and 11 does not impact Purkinje cell degeneration in pcd mice. However, these genes seem to be barely expressed in Purkinje cells (based on the Allen brain atlas and for TTLL6, the authors’ qPCR data) and thus the inability to rescue this pcd phenotype is likely reflective simply of the spatial or cell type differences in expression rather than functional differences, such as differences in substrate specificity. It is possible that these genes may be able to functionally compensate/act like TTLL1 if they were actually expressed in these cells. Similarly, the differential modulation of the olfactory bulb and retinal phenotype may also simply be reflective of the expression level in these cell types rather than differential substrate specificity. The authors should modify their discussion and results section to clarify this, as it may mislead readers.

An interesting experiment, though probably beyond the scope of this study, is to investigate whether transgenic overexpression of TTLL6/11 in Purkinje cells can modulate the phenotype of the pcd/TTLL1 double mutants.

2. As Purkinje cells are a small fraction of the cells in the cerebellum (about 0.3%), qPCR or western blots on whole cerebellar lysate (consisting mostly of granule cells) do not accurately represent molecular changes in these cells. This makes it difficult to correlate molecular changes to phenotypes, for instance the western blots of glutamylation levels in pcd/ttll4 double mutants to the rescue of Purkinje cell loss. The inclusion of immunohistochemistry or in situ hybridization (e.g. RNAScope) for the different members of this family would be helpful for the interpretation of such results. In addition, (semi)-quantitative immunohistochemistry for tubulin glutamylation in the cerebellum would be helpful to determine the role of tubulin polyglutamylation in the neurodegenerative phenotype.

3. The inclusion of microarray data from a timepoint at which most Purkinje cells have already been lost in the pcd mouse adds little meaningful data beyond that shown by the histology as gene expression changes are reflective of the absence of these cells.

Minor suggestions:

1. It is unclear which lobule is shown in the cerebellar histology, or even if the same lobule is shown for the different genotypes (Fig. 2C, 3D etc). This should be clarified, and if possible, a low magnification image of the entire cerebellum should be included.

2. Quantification/statistical analysis should be included for the western blots. In addition, some of the western blots including Fig. 3B and 4B have saturated signal/shadow bands.

3. Do TTLL6/11 affect testes function/male infertility?

4. The authors mention that loss of function mutations in TTLL5 have been shown to cause photoreceptor loss in mice (line 485). Has photoreceptor loss indeed been observed in mouse models? If so, can the authors discuss the discrepancy with their data?

Reviewer #2: In their study, Wu et al. investigate the recently discovered mechanism of hyperglutamylation-induced neurodegeneration. Polyglutamylation is a posttranslational modification (PTM) of tubulin that is highly enriched on microtubules in neurons. Recent work had shown that when this PTM is abnormally accumulated due to the absence of the deglutamylating enzyme CCP1, neurons degenerate. The most emblematic neuron type to degenerate are the cerebellar Purkinje cells.

It had also been shown by several teams that removing one of the key glutamylase enzymes, TTLL1, in the CCP1-KO background prevented the degeneration of the Purkinje cells, demonstrating the causative link between accumulation of polyglutamylation and degeneration.

The question that had so far not been answered is whether other polyglutamylating enzymes are also involved in the degeneration observed in CCP1-KO mice. The current manuscript addresses this question in a systematic manner.

The authors generated knockout mouse models for 6 additional TTLL glutamylase enzymes, and systemically crossed them with the CCP1-KO (pcd) mouse to test their impact on Purkinje-cell degeneration. They find that, as previously shown, TTLL1-KO rescues this phenotype, but rather unexpectedly also TTLL4-KO did so. By contrast, none of the other TTLL enzymes could rescue Purkinje cell degeneration, which was surprising given that some of them are highly expressed in the brain.

Following these exciting results, the authors further show that two other degenerative phenotypes of pcd mice, the degeneration of Mitral cells in the olfactory bulb and the degeneration of the retina are also prevented by knockout of TTLL1 or TTLL4, however, the retina degeneration is only partially protected by TTLL4-KO. By contrast, the male sterility of CCP1-KO mice is not reverted when combined with TTLL1- nor TTLL4-KO.

The crux of these findings is the question why TTLL4-KO protects similar to TTLL1-KO, while other TTLLs have no effect. Most importantly, polyglutamylation, in CCP1-TTLL4-dKO mice is not reduced, while this is the case in CCP1-TTLL1-dKO mice.

The authors have generated an impressive number of combinatorial mouse models to demonstrate the role of each individual TTLL enzyme, which is a remarkable tour de force. Moreover, their finding that only one other glutamylating enzyme, TTLL4 protects neurons from degeneration similar to what had previously shown for TTLL1, while all other enzymes of this family, even those that are highly expressed in the brain, had no effect, is novel, important and exciting. It is therefore unfortunate the in the current form, the manuscript does not value this finding sufficiently. The key problem of this manuscript is its blunt enumeration of experiments the authors have performed, the lack of effort to make figures easy to understand, to guide the reader through their work. Moreover, no attempts to provide quantitative measures for phenotypes have been made, which would be particularly important for the TTLL4/CCP1-dKO model (see below). In its current form, the paper is difficult to read and there are a number of open questions that must be addressed before considering it for publication.

Major points:

1) Fig. 1A: First of all, why did the authors omit TTLL2, TTLL9 and TTLL13 which have been predicted / demonstrated to be glutamylases?

However, the main problem with this figure and the conclusions drawn from it is the fact that it is very difficult to conclude the roles of these enzymes in the tested tissues from these analyses. How can Q-PCR data from different genes be compared? Most likely not at all. What can, however, be compared is the relative expression of the same gene in different organs or tissues. For TTLL6, for instance, it is obvious that it might not play a role in the nervous system, as its expression levels are very low compared to testes. All other enzymes show similar expression levels in brain and testes. To make a convincing point about the specific expression of the enzymes in the nervous system, the authors must compare their expression levels in the brain to the expression levels in other organs, and preferably organs where polyglutamylation plays a less important role (testes are surely not the best control here). Without this comparison, it is hard to tell if the numbers the authors obtained stand for a strong or weak expression.

Finally, it is not clear how many times these analyses were repeated (no statistical analysis of the data). Also, representing the Q-PCR data in the form of diagrams would improve the readability of the figure.

2) Quality of data representation: The paper has some major problems with the quality and the readability of the figures. This is not only a problem for the readability of the manuscript, it might also affect the interpretation of the results. The two main concerns are:

2a) Immunoblots lack molecular weight markers, and are very closely cropped. This is not good practise.

2b) The authors never comment whether they can distinguish alpha- and beta-tubulin in their blots, which would be helpful as they interpret a lot of their results in the light of the enzymatic specificities of the enzymes.

2c) All polyE blots in this manuscript appear to be overloaded, and can therefore not reveal subtle differences in polyglutamylation levels that might occur in some cases. While the previously reported very strong difference between wild type and CCP1-KO (pcd) is visible under these experimental conditions, the authors might have missed a more subtle decrease in polyE signal, for instance in CCP1/TTLL4 dKO mice that show partial Purkinje-cell survival. Being able to see such subtle differences would completely change the interpretation of the paper.

2d) Histology images are not homogenously represented. First, they are so different in terms of colour, and region selected it is not obvious for a reader that the brain regions depicted are comparable. This concerns Fig. 2C, 3D, 4C and 6 (for instance, compare panels in Fig. 2C – they all look completely different). Images need to be better processed, comparable regions should be shown. Brain layers should be labelled. Moreover, given that brain histology is basically the key readout of this work, entire cerebella should be shown additionally to the zoomed-in region.

3) It is not clear why the authors first analyse the TTLL6-KO mice, having shown in Fig. 1A that this enzyme is not expressed in the nervous system? This is counter-intuitive, and becomes particularly perturbing at line 202, where the authors simply conclude that TTLL1, TTLL6 and TTLL11 elongate polyglutamylation on alpha-tubulin, but only TTLL1 appears to contribute to the degeneration of Purkinje cells – citing Fig. 1A. Going to Fig. 1A now, the reader discovers that TTLL6 is not expressed in cerebellum (making the lack of rescue obvious), but TTLL11 is Q-PCR-wise more than 2x more expressed than TTLL1. So how can this be explained? This most likely needs to be put in the context of what Q-PCR data really tell us (see point 1) above).

4) The most striking discovery of this paper is the (partial) rescue the CCP1-related phenotypes by knockout of TTLL4. However, it raises the important question of the underlying mechanism given that in the western blots it seems that TTLL4-KO does not reduce hyperglutamylation, while TTLL1-KO does. As mentioned in point 2c), there might be a slight reduction of polyglutamylation the authors have not detected because of the way they perform their blots, but the question still remains of how TTLL4-KO can rescue given that is does not lower polyglutamylation as does TTLL1-KO. One possible answer could be that in fact all of the observed rescue phenotypes are partial.

Indeed, the authors show that TTLL4-KO can only partially rescue the CCP1-induced degeneration of the retina, but they claim at the same time that it can fully rescue mitral and Purkinje cells based on snapshots of olfactory bulb and cerebellum without any quantification or multiple experiments. Given that in mouse models in which Purkinje cells degenerate only partially, this partial degermation is not homogenous throughout the cerebellum (e.g. Fig. 1G in Liu Y, Lee JW, Ackerman SL (2015) Mutations in the Microtubule-Associated Protein 1A (Map1a) Gene Cause Purkinje Cell Degeneration. J Neurosci 35: 4587-4598), it would be first of all essential to show images of the entire cerebellum (and similarly, the entire olfactory bulb) in order to show that the region the authors have presented currently is representative. Moreover, the authors might attempt to quantify the number of Purkinje cells (and mitral cells) in the different mouse models to determine whether there is a partial degeneration similar to what they see in the retina. Finally, it might be worth checking whether the protective effect of TTLL4 is as efficient as the one by TTLL1 by investigating older mice.

5) The gene expression study in Fig. 5 is awkward: all the genes that the authors find to be downregulated are Purkinje-cell specific genes. So obviously, in all mouse models where the Purkinje cells are lost, the genes specifically expressed in these cells cannot be detected anymore. But this does by no means prove their downregulation, but simply mirrors the loss of Purkinje cells.

Minor points:

1) In the abstract, the authors write “Genetic disruption of polyglutamylation causes neurodegenerative phenotype”. This is misleading, as it could be interpreted as loss of glutamylation (“disruption”). However, what really happens is that accumulating polyglutamylation leads to degeneration. This should be re-formulated.

2) The authors omit the notion that while all glutamylases have a previously demonstrated preference for initiation or elongation, as well as for alpha- or beta-tubulin, these are preferences and not exclusive activities. In other words, one cannot exclude that TTLL1 initiates the glutamate chains it later elongates. They should consider the potential promiscuity of the enzymes when discussing their findings.

3) Fig. 1B: it is important to introduce the reader to polyglutamylation and the involved enzymes. However, this could be done a bit more intuitive than in the current figure that oversimplifies a lot. Why not introducing the notion of alpha- and beta-tubulin preferences into this schematic, and remove it from Fig. 1A, where data are shown?

4) line 182: the authors must explain the connection between PGs1 and TTLL1 mutant mice.

5) in Fig. 7, the zoom does not really reveal what the authors want to show. A more explicit representation should be sought.

6) in Fig. 8, single-channel images as in Fig. 7 would be useful to clearly see the phenotype the authors want to point out. Fig. 7 and 8 could be combined.

7) In the discussion, the authors propose that their observation of the importance of TTLL4 for Purkinje cells might be explained by both, alpha- and beta-tubulin glutamylation participating in this phenotype. However, they did not show that TTLL4 is contributing to beta-tubulin glutamylation in neurons or brain tissue, and they also do not discuss why in this case TTLL7, a known beta-tubulin glutamylase with a very strong expression in brain, and important functions in neurons (Ikegami et al., 2006) does not rescue CCP1-mediated degeneration.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: No: No GEO accession number for microarray dataset provided

Reviewer #2: Yes

**********

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Reviewer #1: No

Reviewer #2: No

25 Jan 2022

Submitted filename: TTLL Revision Response to Comments.docx

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17 Feb 2022

Dear Dr Rong,

Thank you very much for submitting your Research Article entitled 'TTLL1 and TTLL4 polyglutamylases are required for the neurodegenerative phenotypes in pcd mice' to PLOS Genetics.

The manuscript was reviewed again by the original reviewers.  Reviewer #1 was satisfied with the revision, as was, generally, Reviewer #2 but this reviewer identified some aspects of the presentation that need improvement before we can proceed further, including noting some errors as well as suggestions for significantly clarifying the presentation and writing.  We would ask that you look through these suggestions very carefully and ideally make most or all of the changes as we agree it would improve the presentation and thus the impact.

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Reviewer's Responses to Questions

Comments to the Authors:

Please note here if the review is uploaded as an attachment.

Reviewer #1: The authors have addressed my concerns, and the manuscript is much improved. I now support its publication. I would suggest that the authors label the relevant genotypes on the panels of their immunofluorescence figures (Fig3,4,6, 7-9) to make things easier for readers, and avoid labels such as panel Aa''.

Reviewer #2: The authors present a thoroughly revised manuscripts in which they carefully addressed all referees’ comments. As a result, the revised manuscript has hugely improved. Most importantly, the authors have performed a series of new experiments to prove the reproducibility of their observations, which was one of the key concerns in the first round of review. They have further re-focused the story onto the key findings that TTLL1 and TTLL4, but none of the other tested TTLL enzymes play a role in neurodegeneration induced by accumulation of polyglutamylation. This improves the logical flow of the new manuscript, while all important information is still available in the supplementary material.

Given the solidness of this work, it will be a strong contribution to the further mechanistic understanding of the newly emerging role of the tubulin modification polyglutamylation in neurodegeneration. In particular, the observation of the authors that the enzyme TTLL4 contributes to this novel form of degeneration despite the fact that TTLL4 is not the most-strongly expressed TTLL in the brain is intriguing and will help to further elucidate the potentially complex mechanisms leading to the loss of neurons in the future.

Overall, the revised manuscript shows no major weaknesses, experimental data are solid, and results are meticulously discussed. A couple of minor concerns should be addressed before considering the paper for publication:

1) The authors should carefully re-read the manuscript. It appears that there are parts that have not been fully revised, as for instance some figure numbers are wrong: in the discussion they cite Fig 9 for testes, whereas this is Fig 10 in the revised manuscript.

2) In the introduction (first sentence) the authors write that polyglutamylation is involved in neurodegeneration. This is a bold statement, and could mislead the reader into thinking of common neurodegenerative disorders such as Alzheimer’s disease. Specifying that polyglutamylation is involved in a rare condition of childhood degeneration would be more appropriate.

3) In Fig 4A, panel a, the axon tracts stained with Calbindin are less prominent in wildtype as compared to the mutant in panel b. This is most likely related to the choice of the brain section, but could give the misleading impression that wildtype Purkinje cells have a smaller axon tract as compared to the mutant. The authors might consider replacing the image.

4) What makes the manuscript really difficult to read are the figures. There are a number of style issues with the figures, which the authors might want to address:

• Fig 1B: a table packed with so many numbers is far from being easy to read. Why don’t the authors choose to represent their data as graphs?

• Fig 3, 4A, 6, 7, 8, 9 – without a labelling of the panels (genotypes, antibodies used, etc.) these figures are very hard to decipher (one goes forth and back between figure and legend many times before being sure to correctly understand the data). Given that the authors have shown willingness to put such labels in Fig 10, they might want to consider doing so consistently in all figures.

• In most figures showing immunohistochemistry, the authors wrote text labels over the figure panels. Most of these labels are very small and hard to read, and sometimes it is even impossible to read them because of the image below. Bold letters, and perhaps some background shading, could help here. Scale bars are also very thin lines, thus hard to spot. Given that different magnifications are shown side-by-side, it would not heart to directly label the scale bars in the panels.

• Fig 3, 4A and 6 are monochrome images: why do the authors show them in green? They would be much more contrasted in black and white.

• To make Fig 7 and 8 more readable for the non-specialist, a better labelling of the layers, addition of zoom images to show details, plus a schematic representation of the olfactory bulb could be very helpful.

• Fig 2C,D – the authors plot 4 different measurements using very hard-to-distinguish symbols. They might consider the use of color to make these graphs easier to read.

• Fig 4B, 5C,D: the text in these graphs is much too small to be readable.

• Some of the panels showing immunohistochemistry images are very small, in particular Fig 3 and 8. Given the wealth of important information on those images, they could be much larger than for instance an immunoblot with only 4 protein bands in Fig 5.

**********

Have all data underlying the figures and results presented in the manuscript been provided?

Large-scale datasets should be made available via a public repository as described in the PLOS Genetics data availability policy, and numerical data that underlies graphs or summary statistics should be provided in spreadsheet form as supporting information.

Reviewer #1: Yes

Reviewer #2: No: If I am not mistaken, there are no numerical data to the graphs shown in the manuscript, please verify.

**********

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Reviewer #1: No

Reviewer #2: No

22 Feb 2022

Submitted filename: TTLL Revision 1 Response to comments.docx

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24 Feb 2022

Dear Dr Rong,

Thank you very much for submitting your Research Article entitled 'TTLL1 and TTLL4 polyglutamylases are required for the neurodegenerative phenotypes in pcd mice' to PLOS Genetics.

We reviewed your revision and we agree that most of the changes you made are responsive and acceptable.  But in the interest of readability, as the reviewer had been concerned, there are still a few minor changes that we would ask that you make before the manuscript is formally accepted.  Once you address these we will be poised to make the editorial decision very quickly.

First, in principle it is okay if you leave Figure 1b in table form, but it is still an eyeful to look at. Since the gene expression values are whole integers with no decimals, the second decimal place in the SEM is unnecessary and clutters up the table – please use only one decimal place for the SEM values.  Also, please put a space between the expression value and the ± symbol.

Your including genotypes in the figures as the reviewer asked is helpful, but it needs to be clearer still.  It is acceptable to use shorthand gene symbols in the genotypes rather than full formal symbols in the figures, as long as the shorthand is defined somewhere clearly in the text (e.g. Methods).  Thus, pcd should be defined as Agtpbp1 (italicized) pcd (superscript, non italicized, no spaces), and then if you mean homozygous spell that out too (e.g. Agtpbp1 pcd/Agtpbp1 pcd or heterozygous (Agtpbp1 pcd/Agtpbp1 +).  Similar for Ttl1 KO (there is probably an official gene symbol for this KO allele – you can check with JAX if one has been registered; if not, you can use the minus sign to indicate ko, i.e. -).  Also, for pcd specifically in some figures you use pcd and others pcd^3J; please be consistent if you only used one of these.  Last, the double mutant shorthanding is confusing because you are using a slash which is typically reserved for use in genotype copy number; instead of the slash, to indicate double mutants you should use a comma.  That is, if you are going to shorthand Ttl1 mice as Ttl1KO and Agtpbp1 as pcd, and specify in the Methods that these are homozygous, then you could use in the shorthand for the figure: Ttl1KO, pcd).  If there are mixed copy numbers, then that could be further specified, e.g. Ttl1KO/+, pcd/pcd.

Again, once these issues are addressed we will finalize a decision rapidly.

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Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

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Wayne N. Frankel

Associate Editor

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Gregory Barsh

Editor-in-Chief

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8 Mar 2022

Submitted filename: Editor comments to the authors.docx

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14 Mar 2022

Dear Dr Rong,

We are pleased to inform you that your manuscript entitled "TTLL1 and TTLL4 polyglutamylases are required for the neurodegenerative phenotypes in pcd mice" has been editorially accepted for publication in PLOS Genetics. Congratulations!

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Comments from the reviewers (if applicable):

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8 Apr 2022

PGENETICS-D-21-00374R3

TTLL1 and TTLL4 polyglutamylases are required for the neurodegenerative phenotypes in pcd mice

Dear Dr Morgan,

We are pleased to inform you that your manuscript entitled "TTLL1 and TTLL4 polyglutamylases are required for the neurodegenerative phenotypes in pcd mice" has been formally accepted for publication in PLOS Genetics! Your manuscript is now with our production department and you will be notified of the publication date in due course.

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