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Current Neurobiology

Dynamic expression of SynDIG1 mRNA in cerebellar Purkinje neurons

Author(s): Elva Díaz

Vol. 1, No. 1 (2010-01 - 2010-06)

Current Neurobiology 2010; 1 (1): 77-81

Elva Díaz

Department of Pharmacology, UC Davis School of Medicine, Davis, CA 95616, USA

Abstract

During development of the central nervous system, successive intrinsic and extrinsic programs control the specification, proliferation, and migration of individual neuronal cell types that ultimately result in the formation of precise synaptic connections. In previous research, expression profiling of the developing mouse cerebellum identified genes expressed during specific phases of neuronal differentiation. Based on this study, a novel type II transmembrane protein (SynDIG1) that regulates AMPA receptor content at developing synapses was discovered. Here I provide a detailed analysis of SynDIG1 mRNA expression in the developing mouse cerebellum. SynDIG1 mRNA is expressed exclusively in Purkinje neurons as demonstrated by analysis of Lurcher and weaver mutant mice in which Purkinje neurons or granule neurons degenerate, respectively. SynDIG1 mRNA expression is most prominent in mouse cerebellum at postnatal day 14, the peak of synaptogenesis in rodents. Interestingly, SynDIG1 mRNA appears to localize to Purkinje neuron dendrites, suggesting the intriguing possibility that SynDIG1 mRNA is subject to local protein synthesis at synapses. Taken together, these results demonstrate that SynDIG1 mRNA expression is highly dynamic with restricted expression to Purkinje neurons in the developing mouse cerebellum.

Introduction

The cerebellar cortex is a popular model system for the analysis of neuronal differentiation due to the simplicity of its cellular architecture. The cerebellum is composed of a small number of morphologically and molecularly distinct neuronal cell types (see [1] for review). Mature granule neurons extend multiple short dendrites that receive synaptic input from mossy fiber afferents and Golgi neurons, whereas granule neuron axons (the parallel fibers of the cerebellum) synapse on distal dendritic spines of Purkinje neurons. The proximal dendrites of Purkinje cells are innervated by multiple synapses from a single climbing fiber originating from the inferior olive.

During development of the cerebellum, Purkinje neurons arise from the ventricular zone along the lining of the dorsal aspect of the fourth ventricle (see [2] for review). Purkinje neurons become postmitotic during embryonic development and migrate along radial glia fibers to form a multi-cell layer below the external granular layer (EGL). Beginning at birth, Purkinje neuron dendrites undergo dynamic remodeling based on intrinsic and extrinsic cues [3]. The Purkinje neuron dendritic arbor is mature by postnatal day 13 (P13); however, Purkinje neuron dendrites undergo dynamic synapse remodeling events until P30. For example, at birth, each Purkinje neuron is innervated by multiple climbing fibers of the inferior olive nuclei; however, these surplus climbing fibers are eliminated during postnatal development, such that by P20 mono-innervation is attained.

An attractive aspect of the cerebellum as a model system is the large number of mouse mutants that affect selectively Purkinje neuron or granule neuron development. For example, in heterozygous Lurcher (Lc) mice, Purkinje neurons degenerate in the second postnatal week of development due to a point mutation in the δ2 glutamate receptor [4], which is selectively expressed in cerebellar Purkinje neurons [5]. Subsequently, a dramatic loss of granule neurons is observed [6]. In weaver (wv) mice, granule neurons die in the EGL due to expression of a mutant potassium channel in granule neurons [7], resulting in greatly reduced numbers of mature granule neurons in the internal granule layer (IGL) [8, 9]. This in turn results in defects in Purkinje neuron development and aberrant morphologies of Purkinje neuron dendrites [10, 11].

Previously, a DNA microarray approach was applied to expression profile the cerebellum in wild type and mutant mouse lines with defects in neuronal differentiation [12]. This approach identified a novel transmembrane protein (SynDIG1) that regulates excitatory synapse development via interaction with AMPA receptors [13]. In wild type cerebellum, SynDIG1 mRNA is upregulated during postnatal development; in contrast, SynDIG1 upregulation is defective in Lc cerebellum [12]. In situ hybridization with SynDIG1 digoxigenin-labeled riboprobes in mouse brain sections demonstrated that SynDIG1 mRNA is expressed in Purkinje neurons in cerebellum as well as throughout the hippocampus [13]. Here I provide a detailed analysis of SynDIG1 mRNA expression in the developing mouse cerebellum using the more sensitive radioactive in situ hybridization method. Consistent with the microarray expression profiling study [12], SynDIG1 mRNA is expressed exclusively in Purkinje neurons as demonstrated by analysis of Lc and wv mutant mice in which Purkinje neurons or granule neurons degenerate, respectively. SynDIG1 mRNA is expressed in immature Purkinje neurons; however expression is most prominent at P14, during the peak of synapse development in rodents. Interestingly, SynDIG1 mRNA appears to localize to both Purkinje neuron cell bodies and dendrites, suggesting the intriguing possibility that SynDIG1 mRNA is subject to local protein synthesis at synapses. Taken together, these results demonstrate that SynDIG1 mRNA expression is highly dynamic with restricted expression to Purkinje neurons in the developing mouse cerebellum.

Materials and Methods

Animals

CD-1 mice were purchased from Charles River. Breeder pairs for mutant mouse lines were purchased from the Jackson Laboratory. All mice were bred and maintained in the animal facility at UC Davis. Weaver homozygotes (wv/wv), heterozygotes (wv/+) and wild-type littermates were generated by mating B6CBACa-A w-J/A –Kcnj6 wv mice. Genotypes were determined by a restriction site-generating PCR protocol [18]. Lurcher heterozygotes (Lc/+) and wild-type littermates were generated by mating B6CBACa-A w-J/A –Grid2 Lc male mice and wild-type females; genotypes were determined by restriction fragment length polymorphism analysis [4] and/or behavior for adult animals. The use and maintenance of animals were according to the guidelines set forth by UC Davis, NIH, and AALAC.

In situ hybridization

In situ hybridization on fresh frozen sections was performed as described [19]. Briefly, SynDIG1 cDNA sequence (Riken AV149920) was used as template for in vitro transcription to produce anti-sense 35 S-UTP labeled probes. Probes were purified on S-200 micro-spin columns (Amersham) and diluted to 8×108 cpm/ml in Wilkinson’s hybridization buffer [20]. Fresh frozen CD-1 brains were cut into 20 μm sections on a cryostat. Sections were fixed in 4% paraformaldehyde / 1X phosphate buffered saline (PBS) for 10 min, washed in PBS, acetylated for 10 min, washed in PBS, and incubated with Wilkinson’s hybridization buffer for 4 hr. RNA probes were applied evenly by “painting” with parafilm. Slides were coverslipped and incubated in a humidified chamber overnight at 65°C. The next day, slides were washed in 0.2 X SSC, 10 mM DTT at 72°C for 4×30 min (100 mM DTT in the first wash). Slides were cooled in RNase buffer (0.5 M NaCl, 10 mM Tris pH 7.5, 5 mM EDTA), incubated with 5 μg/ml RNaseA for 30 min at 37°C, washed 2×30 min with RNase buffer, 4×15 min with 0.2 X SSC at 72°C, and dehydrated through an ethanol series with 0.3 M NH4OAc. Slides were coated with NTB2 emulsion (Kodak) and exposed for 10-14 days. Slides were developed with D19 developer (Kodak), rinsed with water and fixed at 15°C. Slides were dehydrated through an ethanol series, incubated with xylenes and mounted. Images were taken with a digital camera mounted on a Zeiss Axioplan2 upright microscope under dark-field conditions.

Results and Discussion

SynDIG1 mRNA is expressed in Purkinje neuron cell bodies and dendrites

To examine the distribution of SynDIG1 mRNA in finer detail, in situ hybridization with 35S-UTP-labeled riboprobes was performed with CD-1 mouse brain sections at different ages (Figure 1). As previously demonstrated with digoxigenin-labeled riboprobes, SynDIG1 mRNA is expressed in Purkinje neurons in cerebellum [13]. SynDIG1 mRNA expression is detected as early as P6 in immature Purkinje neurons throughout the cerebellar cortex (Figure 1A). At P6, immature Purkinje neurons are practically devoid of dendrites [see [3] for review]. Thus, as expected, examination of higher magnification images revealed that SynDIG1 mRNA expression is restricted to Purkinje neuron cell bodies (Figure 1B).

SynDIG1 mRNA expression is most prominent at P14 in mouse cerebellum (Figure 1C), consistent with its peak of protein expression in whole brain extracts [13]. Strikingly, examination of higher magnification images revealed that SynDIG1 transcripts are detectable in Purkinje neuron cell bodies and dendrites within the molecular layer (Figure 1D). By P14, the dendrites of young Purkinje neurons exhibit all features of adult Purkinje neurons [see [3] for review]; however, Purkinje neuron dendrites undergo dynamic synapse remodeling events until P30. At P22, SynDIG1 transcripts are still present in Purkinje neuron cell bodies (Figure 1E); however, examination of higher magnification images revealed that SynDIG1 mRNA expression in dendrites was significantly reduced compared with P14 cerebellum (compare Figures 1F and 1D). Thus, while SynDIG1 mRNA expression is maintained in Purkinje neuron cell bodies throughout development, SynDIG1 mRNA expression in dendrites peaks during the period of Purkinje neuron synaptogenesis.

The observation that SynDIG1 mRNA expression is most prominent during the peak of synaptogenesis in rodents in consistent with the previous demonstration that SynDIG1 functions to regulate AMPA receptor content at developing synapses [13]. SynDIG1 protein is present in adult brain but at a reduced level compared with P14 brain [13].

Thus, SynDIG1 mRNA abundance dictates SynDIG1 protein level. Intriguingly, SynDIG1 transcripts are detected in the molecular layer of the cerebellum, the location of Purkinje cell dendrites, suggesting that SynDIG1 mRNA might be subject to local mRNA translation at synapses. Indeed, local protein synthesis is thought to underlie aspects of synaptic plasticity and higher order cognitive function (see [14] for review). However, SynDIG1 mRNA expression in dendrites is enriched in P14 brain, suggesting that local translation of SynDIG1 mRNA might occur only at nascent synapses.

Figure 1. SynDIG1 mRNA expression in the developing mouse cerebellum.

Sagittal sections of mouse cerebellum at postnatal day 6 (A, B), postnatal day 14 (C, D), and postnatal day 22 (E, F) were hybridized in situ with antisense 35 S-UTP labeled probe for SynDIG1. Note that SynDIG1 mRNA expression is most prominent at P14 in Purkinje neuron cell bodies and dendrites. Scale bar, 400 μm (A, C, E), 100 μm (B, D, E).

Figure 2. SynDIG1 mRNA is expressed selectively in cerebellar Purkinje neurons. Sagittal sections of cerebellum at postnatal day 28 from wild type mice (A, B) and Lurcher heterozygous littermates (C, D) were hybridized in situ with antisense 35 S-UTP labeled probe for SynDIG1. Note that SynDIG1 mRNA expression is absent in Lc/+ cerebellum compared with wild type littermates. Scale bar, 400 μm (A, C, E), 100 μm (B, D, E).

Figure 3. SynDIG1 expression in Purkinje neurons does not require synaptic input from granule neurons Sagittal sections of cerebellum at postnatal day 25 from wild type mice (A, B), weaver heterozygous littermates (C, D), and weaver homozygous littermates (E, F) were hybridized in situ with antisense 35 S-UTP labeled probe for SynDIG1. Note that SynDIG1 mRNA expression in dendrites but not cell bodies is dependent on the presence of granule neurons. Scale bar, 400 μm (A, C, E), 100 μm (B, D, E).

SynDIG1 mRNA expression is restricted to Purkinje neurons in cerebellum

To conclusively prove that SynDIG1 is expressed in Purkinje neurons and not radial glial cells that are also present in the Purkinje cell layer [1], mouse cerebellum sections from Lc/+mice and wild type littermates were examined with in situ hybridization (Figure 2). In Lc/+ mice, there is massive Purkinje neuron death during the second postnatal week of development due to a point mutation in the δ2 glutamate receptor [4], which is selectively expressed in cerebellar Purkinje neurons [5]. As expected, SynDIG1 transcripts are present in Purkinje neuron cell bodies in cerebellar sections from wild type mice (Figures 2A-2B). In contrast, SynDIG1 transcripts are absent in cerebellar sections derived from Lc/+ littermates (Figure 2C). Indeed, examination of higher magnification images of Lc/+ cerebellum did not reveal any significant labeling above background levels (Figure 2D). Thus, SynDIG1 mRNA is expressed selectively in Purkinje neurons in the mouse cerebellum.

SynDIG1 mRNA expression does not require granule neuron synaptic input

The development of Purkinje neurons is affected by bi-directional cell-cell interactions with granule neurons [15]. To test if SynDIG1 expression in Purkinje neurons is dependent on the presence of granule neurons, cerebellar sections from wv mutant mice were examined with in situ hybridization (Figure 3). In homozygous wv/wv mice, granule neuron precursors are correctly specified and proliferate in the EGL; however, the post-mitotic cells die before they migrate to the IGL [10, 16].

As expected, SynDIG1 transcripts are present in Purkinje neuron cell bodies in cerebellar sections from wild type mice (Figures 3A, 3B). In addition, SynDIG1 transcripts are present in cerebellar sections derived from wv/+ littermates (Figures 3C, 3D) and wv/wv littermates (Figures 3E, 3F). Indeed, examination of higher magnification images of wv/+ and wv/wv cerebellum revealed that although Purkinje neurons are disordered in these mutant mice, SynDIG1 mRNA expression is detectable in Purkinje neuron cell bodies (Figures 3D, 3F). In contrast, SynDIG1 transcripts are absent from Purkinje neuron dendrites in wv/wv mice (Figure 3F). Thus, while SynDIG1 expression in Purkinje neuron cell bodies does not require synaptic input from granule neurons, SynDIG1 expression in Purkinje neuron dendrites is dependent on the presence of granule neurons.

The observation that SynDIG1 mRNA expression in Purkinje neuron dendrites but not cell bodies requires synaptic input from granule neurons suggests that SynDIG1 mRNA is transported to developing synapses via retrograde signaling from presynaptic parallel fibers of the granule neurons. Early events in synapse development include clustering of synaptic vesicles to the presynaptic active zone, and NMDA receptors to the postsynaptic density while later events include clustering of AMPA receptors and synaptic activity directs whether synapses will be stabilized, eliminated or strengthened (see [17] for review). SynDIG1 regulates the content of AMPA receptors but not NMDA receptors at developing excitatory synapses [13], suggesting that SynDIG1 mRNA translation at nascent synapse might provide an immediate signal to trigger synapse stabilization via the incorporation of synaptic AMPA receptors. Further experiments will be necessary to test this interesting possibility.

In summary, SynDIG1 mRNA is expressed exclusively in Purkinje neurons and is most prominent at P14, the peak of synaptogenesis in rodents. Interestingly, SynDIG1 mRNA appears to localize to Purkinje neuron dendrites, suggesting the intriguing possibility that SynDIG1 mRNA is subject to local protein synthesis at synapses. Furthermore, SynDIG1 mRNA localization to Purkinje neuron dendrites but not cell bodies is dependent on the presence of granule neurons. Taken together, these results demonstrate that SynDIG1 mRNA expression is highly dynamic with restricted expression to Purkinje neuron cell bodies and dendrites in the developing mouse cerebellum.

Acknowledgments

This work was supported by grants to E.D. from the Alfred P. Sloan Research Foundation, the Whitehall Foundation, and the National Science Foundation (0542281; 0842724).

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Correspondence: ediaz(at)ucdavis.edu

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