Spatiotemporal Distribution of VPS13A in the Mouse Brain

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1		Manuel J Rodriguez	+ 2039 word(s)	2039	2021-12-07 04:27:50

This entry is adapted from the peer-reviewed paper 10.3390/ijms222313018

Loss-of-function mutations in the human vacuolar protein sorting 13 homolog A (VPS13A) gene cause Chorea-acanthocytosis (ChAc). As very little is known about the VPS13A expression in the brain, The main objective of this work was to assess for the first time the spatiotemporal distribution of VPS13A in the mouse brain. Understanding the distinct expression pattern of VPS13A provides relevant information to unravel pathophysiological hallmarks of ChAc.

Chorea-acanthocytosis VPS13A brain distribution movement disorders basal ganglia neurodegeneration

1. Introduction

The human vacuolar protein sorting the 13 homolog A (VPS13A) gene encodes a large protein of 3174 amino acids named VPS13A or chorein. Human VPS13A protein is of great interest because loss-of-function mutations in its coding gene lead to Chorea-acanthocytosis (ChAc; MIM 200150), a very rare and complex autosomal recessive adult-onset neurodegenerative disorder^[1]. In accordance with this etiology, ChAc has recently proposed to be renamed as VPS13A disease^[2]. The main neuropathologic feature in VPS13A disease is a selective degeneration of the caudate and putamen nuclei^[3]^[4], due to massive cell death of medium spiny neurons and striatal interneurons^[5]. Moreover, many other neuronal subtypes, such as dopaminergic neurons or motoneurons, are affected as well, contributing to the explanation of a plethora of pathological symptoms that include chorea, dystonia, involuntary oral biting, and orofacial dyskinesia, among others^[6].

VPS13A is widely distributed in the body. According to the human Genotype-Tissue Expression (GTEx) project (Accession number: ENSG00000197969.11; October 2021), VPS13A is expressed by many tissues including in the testis and kidney, as well as cardiovascular and digestive tissues. A similar distribution pattern was found in the mouse ^[7]. Nevertheless, little is known about the VPS13A distribution in neural cells and the brain. A preliminary study showed that VPS13A is present in microsomal and synaptosomal fractions in the mouse brain^[7]. In the same study, cell-specific patterns of VPS13A-like immunoreactivity were detected in the striatum, cerebral cortex, and hippocampus^[7]. Despite that general overview, a time course of VPS13A expression and an in-depth, detailed protein brain localization were necessary. Moreover, it is interesting to know when VPS13A expression starts for understanding a putative role of the protein in development. Therefore, a time course and regional expression analysis of VPS13A in the mouse brain is valuable in assessing the earliest possible influence of the lack of VPS13A in ChAc pathogenesis.

2. VPS13A Expression Starts from Embryonic Stage and Is Stable over Time

To assess the time course of VPS13A expression in the mouse brain, the authors performed fluorescent in situ hybridization (FISH) and quantitative real-time PCR (qRT-PCR) of the cerebral cortex, striatum, hippocampus, and cerebellum at different ages (E15.5, P0, P7, P34 and 16 weeks). At the mid-stages of mouse brain development (E15.5), we found that VPS13A is predominantly expressed in neuron-enriched areas compared with germinal zones in both the cortex and hippocampus. This finding is consistent with single-cell RNA profiling data from E15.5 mouse cortex^[8], which also showed that VPS13A is highly expressed in neurons compared with apical progenitors. We then analyzed the VPS13A expression in P0, P7, and P34 postnatal stages to evaluate putative changes of expression and/or distribution over time. We found VPS13A expression in the cerebral cortex, striatum, hippocampus, and cerebellum for all ages analyzed. That labeling was homogeneous throughout all layers of the cerebral cortex, the striatum, and the pyramidal layer of all hippocampal subfields as well as the granular layer of dentate gyrus.

3. Overall Distribution of VPS13A mRNA and Protein in the Adult Mouse Brain

To further assess the distribution of VPS13A mRNA and protein in the adult mouse brain, authors performed FISH and immunohistochemistry procedures, respectively, and evaluated the pattern of expression throughout the brain (Table 1). They found a wide distribution of mRNA and protein labeling throughout the mouse brain, with distinct staining intensity profiles between nuclei. In general, the mRNA localization resembled that of the protein one, with minor changes in intensity profile.

Table 1. VPS13A expression in the mouse brain.

Brain Structure	mRNA	Protein	Brain Structure	mRNA	Protein
Motor Cortex			Basal ganglia
Layer I	−	−	Caudate putamen	+	+
Layer II/III	++	++	Fundus of striatum	+	+
Layer V	++	++	Globus pallidus	+	+
Layer VI	++	++	Bed nucleus of stria terminalis	++	+
Somatosensory Cortex			Nucleus accumbens	+	+
Layer I	−	−	Substantia nigra	+	+
Layer II/III	++	+	Subthalamic nucleus	+++	++
Layer IV	++	+	Amygdaloid complex
Layer V	+++	++	Basolateral amygdalar nucleus	++	++
Layer VI	++	+	Basomedial amygdalar nucleus	++	++
Visual Cortex			Central amygdalar nucleus	++	++
Layer I	−	−	Thalamus
Layer II/III	++	+	Reticular nucleus	++	++
Layer IV	++	+	Lateral dorsal nucleus	+	+
Layer V	++	++	Posterior complex	+	+
Layer VI	++	+	Ventral medial nucleus	+	+
Entorhinal Area			Ventral/Dorsal geniculate n.	+	+
Layer I	−	−	Paraventricular nucleus	++	++
Layer II	+++	+++	Medial habenula	++	++
Layer III	++	+	Nucleus of reuniens	+	+
Layer IV	++	+	Hypothalamus
Layer V/VI	++	+	Paraventricular hypothalamic n.	++	++
Hippocampal Region			Ventromedial hypothalamic n.	++	++
CA3	+++	++	Ventral premammillary nucleus	++	++
CA2	+++	++	Lateral mammillary nucleus	++	n/a
CA1	++	+	Medial mammillary nucleus	++	n/a
Granular layer of the DG	+++	+	Medial preoptic area	+	+
Polymorphic layer of the DG	+	+	Arcuate hypothalamic nucleus	+	+
Molecular layer of the DG	−	−	Suprachiasmatic nucleus	+	+
Hilus	++	++	Zona incerta	+	+
Postsubiculum	++	++	Septal region
Presubiculum	++	++	Lateral septal nucleus	+	+
Subiculum	++	+++	Medial septal nucleus	+	+
Induseum griseum	++	++	Septohippocampal nucleus	+	+
Mid-Brain			Cerebellum
Superior colliculus	+	+	Purkinje cell layer	+++	+++
Inferior colliculus	+	+	Molecular layer	+	+
Edinger–Westphal nucleus	++	++	Granular layer	++	++
Trochelar nucleus	++	n/a	White matter structures
Oculomotor nucleus	++	n/a	Corpus callosum	+	−
Pons			Anterior commissure	−	−
Pontine gray	+++	+++	Fornix system	+	+
Tegmental reticular nucleus	+++	+++	Optic tract	+	−
Pontine reticular nucleus	++	n/a	Ventral hippocampal commissure	+	−
Motor nucleus trigeminal	+++	n/a	Non-neuronal tissue
Medulla			Choroid plexus	+++	+++
Gigantocellular reticular n.	++	++
Nucleus raphe magnus	++	++
Facial motor nucleus	+++	n/a

Relative expression levels of VPS13A mRNA and protein in adult mouse brain are expressed in the following four categories: − not detectable, + weak signal, ++ moderate signal, and +++ strong signal. CA, Cornu Ammonis; DG, dentate gyrus; n., nucleus.

Authors observed VPS13A mRNA and protein throughout Layers II to VI of the cerebral cortex, with distinct intensity profiles between different cortical regions (Table 1). Thus, the motor cortex presented the strongest VPS13A labeling. Conversely, the somatosensory cortex had moderate staining. At the cellular level, VPS13A immunostaining was mainly located in the perinuclear zone. Positive staining was also found in the apical dendrite of pyramidal neurons. In the basal ganglia nuclei, cells from the caudate putamen, globus pallidus, and substantia nigra had the weakest VPS13A labeling . Within the thalamic nuclei, the reticular and paraventricular nuclei presented the higher VPS13A expression, compared with the other thalamic nuclei, which presented moderate labeling. Finally, the subthalamic nucleus presented high VPS13A mRNA staining and moderate protein labeling.

VPS13A expression was also evaluated in hippocampus-related structures, including input and output nuclei and moderate staining was observed in the entorhinal cortex (Table 1). The hippocampal formation presented high VPS13A labeling in the pyramidal layer of all hippocampal subfields and the granular layer of dentate gyrus, with a more intense mRNA labeling in CA3 and CA2 subfields and dentate gyrus. However, there were differences between FISH and immunohistochemical labelings (Table 1). Thus, VPS13A protein staining was moderate in the CA1 pyramidal layer and high in the CA3 and CA2 pyramidal layers, whereas the staining in the granular dentate gyrus was considerably weaker.

Moderate VPS13A expression was found in hypothalamic regions. The septal nucleus also presented moderate VPS13A expression. The gigantocellular reticular nucleus and the nucleus raphe magnus also presented high VPS13A mRNA and protein staining, compared with the other medullar nuclei (Table 1). Finally, the pons was one of the structures that presented the highest VPS13A expression (Table 1). The pontine gray, tegmental reticular, and pontine reticular nuclei were the subnuclei of the pons with the highest mRNA and protein labeling. The cerebellum was another of the nuclei with high VPS13A expression. Particularly, the Purkinje cells displayed the highest labeling in this brain region, while cells in the granular layer presented moderate VPS13A expression and the molecular layer presented the weakest labeling (Table 1).

4. VPS13A Is Enriched in Glutamatergic, GABAergic, and Cholinergic Neurons

The expression pattern of VPS13A suggests that it is expressed in glutamatergic neurons, as observed in the cerebral cortex . It is also expressed by GABAergic neurons, as observed in Purkinje cells in the cerebellum (Table 1), indicating that VPS13A could be expressed in different neuronal types. The authors then analyzed the expression of VPS13A in different neuronal subpopulations by double immunostaining using antibodies against VPS13A and specific markers for GABAergic neuronal subpopulations or cholinergic neurons. They found VPS13A immunostaining in calbindin-positive GABAergic neurons, in parvalbumin-positive GABAergic neurons, and in ChAT-positive cholinergic neurons. To further evaluate whether glial cells also express VPS13A, they carried out double immunostaining using antibodies against this protein and either specific markers for astrocytes, oligodendrocytes, or microglia, respectively. Also, VPS13A staining was found in some, but not all, GFAP-positive cells. Finally, VPS13A staining was undetected in neither CNPase-positive oligodendrocytes nor Iba1-positive microglia.

5. VPS13A Is Present Mainly in the Soma of Neurons and Co-Localizes with Both Endoplasmic Reticulum and Mitochondria

These brain tissue observations indicate that the VPS13A protein is expressed in the soma of neurons. VPS13A immunolabeling was also present in neuronal processes, such as pyramidal apical dendrites. Analysis of the subcellular VPS13A distribution in mouse cortical primary cultured neurons showed that cultured neurons present a strong punctate VPS13A labeling in the perinuclear zone, followed by punctate staining of lower intensity in neuronal processes, whereas the nucleus was devoid of specific staining. VPS13A has been described to interact with endoplasmic reticulum and mitochondria in yeast. Authors thus evaluated if these interactions were also present in neuronal cells, by carrying out double immunostaining in cortical primary cultures using antibodies against VPS13A, plus either specific marker for endoplasmic reticulum membrane or external mitochondrial membrane. VPS13A labeling co-localized with both organelle markers, especially with the endoplasmic reticulum one, suggesting stronger enrichment in this compartment in neurons.

To investigate whether VPS13A is present, not only in the dendritic shaft but also in the synaptic terminals, the authors assessed its presence in synaptic spines of cultured cortical neurons by double staining using phalloidin to label dendritic F-actin puncta. They found weak VPS13A immunolabeling within dendritic spines of cortical primary cultures. This was consistent with the VPS13A presence in isolated crude synaptosomes of cerebral cortex tissue. Authors found VPS13A present but not significantly enriched in the crude synaptosome fraction of cerebral cortex tissue.

6. VPS13A Neuroanatomical Distribution and Chorea-Acanthocytosis

Characterizing the detailed VPS13A mRNA and protein neuroanatomical distribution help to unravel its function in the brain and provide novel insights toward the knowledge of ChAc pathophysiology. VPS13A is a stable protein expressed heterogeneously throughout distinct mouse brain nuclei and its expression pattern can provide the basis for future studies aiming to further understand the pathophysiological hallmarks of the VPS13A disease. While VPS13A subcellular localization suggests that it is not directly involved in the core molecular mechanisms of synaptic transmission, VPS13A may have a role in maintaining neuronal homeostasis and function. Indeed, the VPS13A distinct brain distribution contributes to explain the ChAc neuropathology. For example, severe atrophy, neuronal loss and gliosis have been found in the hippocampus, temporal and frontal lobes, or prefrontal cortex of some patients^[4]^[9]^[10]. Other authors report cerebellar atrophy^[11], impairment of the hypothalamic endocrine function^[12], and oculomotor abnormalities due to brainstem dysfunction^[13]. Thus, the high to moderate VPS13A expression levels in these brain areas evidences the important role of this protein in neuronal functioning and survival along the nervous system. However, the main neuropathological feature in ChAc patients is the selective degeneration of the caudate nucleus and putamen^[14] and at a lower extend other basal ganglia nuclei^[3]. Interestingly, weak VPS13A staining was observed in these basal ganglia nuclei. Thus, the vulnerability of striatal neurons to VPS13A disease seems not to be related to the amount of protein present in the cell, but to specific striatal functional properties and medium spiny neuron cell processes specifically affected by the lack of VPS13A. Nevertheless, this detailed VPS13A brain distribution maps of mRNA and protein is the first step to unravel the function of VPS13A in neurons and should help to characterize its role in the basal ganglia brain circuitry to finally understand the ChAc neuropathology.

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Manuel J Rodriguez

Esther García-García

Nerea Chaparro Cabanillas

Mercè Masana

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Entry Collection: Neurodegeneration

Update Date: 14 Jan 2022

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