Immunological studies on the Purkinje cells from rat and mouse cerebella. I. Evidence for antibodies characteristic of the Purkinje cells.

Antibodies have been raised against an enriched preparation of isolated rat cerebellar Purkinje cells. The immunoglobulins were labeled with ¹²⁵I and the strength and specificity of the serum determined by a direct binding assay on cerebellar membranes. About 2% of the ¹²⁵I-labeled IgG bound to an excess of cerebellar membranes. Absorption with heart and liver membranes removed 80.5% of the ¹²⁵I-labeled IgG binding to cerebellar membranes; absorption with cerebrum membranes removed 13% more; the remaining 6.5% were directed specifically against cerebellar membranes. An enriched ¹²⁵I-labeled anti-Purkinje antibody population was prepared by absorption and subsequent elution from cerebellar membranes. The absorption pattern with heart, liver, and cerebrum membranes resembled that found with the total population of IgG except that the nonspecific binding was significantly diminished. The Purkinje cell degeneration (pcd) mouse mutant was used to assess the specificity of the serum toward the Purkinje cells. After absorption of the enriched anti-Purkinje antibodies with heart, liver, and cerebrum membranes, the binding of labeled IgG to membranes prepared from pcd/pcd cerebella was about one-fourth that found with control cerebella. The direct immunoperoxidase technique performed on smears of purified Purkinje and granule cells shows that the unabsorbed serum stains both classes of cells, but that after absorption with liver, heart, and cerebrum membranes, only the Purkinje cells react positively.     

Loss of Sulfoglucuronyl and Other Neolactoglycolipids in Purkinje Cell Abnormality Murine Mutants

A significant reduction in the content of two members of the sulfoglucuronyl-neolacto series of glycolipids (SGGLs), 3-sulfoglucuronyl-lacto-N-neotetraosylceramide (SGGL-1) and 3-sulfoglucuronyl lacto-N-norhexaosylceramide (SGGL-2), in the cerebellum of the Purkinje cell abnormality mutants, Purkinje cell degeneration (pcd/pcd), lurcher (Lc/+), and staggerer (sg/sg), was also confirmed in the mildly affected nervous (nr/nr) mutant. The expression of SGGLs was studied during development of the pcd/pcd mutant cerebellum, and it was shown that the rate of decline in the level of SGGLs practically coincided with the loss of Purkinje cell perikarya. This indicated that SGGLs are primarily localized in Purkinje cells and that initially, at least, there is no genetic defect in the biosynthesis of SGGLs in the mutant. The precursors of SGGLs, viz., lacto-N-neotetraosylceramide (paragloboside) and lacto-N-norhexaosylceramide, as well as other glycolipids derived from these precursors, such as X-determinant fucoglycolipids and disialosyllacto-N-neotetraosylceramide, were also present in normal cerebellum. Levels of paragloboside and its other derivatives, similar to SGGLs, were also significantly reduced in the Purkinje cell abnormality mutants pcd/pcd, sg/sg, Lc/+, and nr/nr but were normal in other cerebellar mutants, such as quaking (qk/qk), weaver (wv/wv), and reeler (rl/rl), where Purkinje cells are not involved. Thus, the entire paragloboside family of glycolipids is primarily associated with Purkinje cells in the cerebellum. Although levels of monoclonal antibody HNK-1-reactive glycolipids were reduced in the Purkinje cell abnormality mutants, HNK-1-reactive glycoproteins were not affected in these mutants.     

Developmental expression and intracellular location of P400 protein characteristic of Purkinje cells in the mouse cerebellum

The developmental expression and intracellular localization of a cerebellum-characteristic 250-kDa glycoprotein, P400 protein, were studied by immunohistochemical and immunoblot methods using a monoclonal antibody against P400 protein. In the cerebellum of normal mouse, the expression of P400 protein increased from Postnatal Day 3 to Day 21. This enhancement of P400 protein expression occurred only in the Purkinje cells and proceeded with the growth of their dendritic arborization. Electron microscopic analysis indicated that P400 protein is present at the plasma membrane, the endoplasmic reticulum, and the postsynaptic densities of Purkinje cells. Immunohistochemistry of the cerebella of neurological mutant mice indicated that the Purkinje cells of reeler, weaver, and pcd mutant mice retain the ability to produce a large amount of P400 protein. However, the Purkinje cells of staggerer mutant mouse proved to be incapable of enhanced P400 protein expression. These results indicate that P400 protein is a Purkinje cell-characteristic plasma membrane-associated glycoprotein, which is also present at the postsynaptic density and endoplasmic reticulum and that the expression of P400 protein in Purkinje cells is closely associated with the growth of their dendritic arborization.     

Differential Cellular Enrichment of Gangliosides in the Mouse Cerebellum: Analysis Using Neurological Mutants

Abstract: The cellular distribution of gangliosides in the cerebellum was studied in a series of adult mouse mutants that lose specific populations of neurons. The weaver ( wv ) mutation destroys the vast majority of granule cells, whereas the Purkinje cell degeneration mutation ( pcd ) destroys the vast majority of Purkinje cells. The staggerer ( sg ) and lurcher ( Lc ) mutations, on the other hand, destroy the vast majority of both granule and Purkinje cells. A proliferation of reactive glial cells, which occurs as a consequence of neuronal loss, has been reported in the sg/sg and pcd/pcd mutants, but not in the wv/wv mutant. Compared with the normal (+/+) mice, the concentration (μg/100 mg dry weight) of G_D1a was significantly reduced in those mutants that lost granule cells, but was not reduced in the pcd/pcd mutant. The concentration of G_TIa, on the other hand, was significantly reduced in those mutants that lost Purkinje cells, but was not reduced in the wv/wv mutant. A significant elevation in the concentration of G_D3, which may be related to the proliferation of reactive glial cells, was observed in the pcd/pcd, sglsg , and Lc /+ mutants, but was not observed in the wv/wv mutant. Because these ganglioside abnormalities were confined to the cerebellum, they cannot result from genetic defects in ganglioside metabolism. Instead, these abnormalities result from a differential enrichment of gangliosides in neural membranes. Our findings suggest that G_DT1a is more heavily concentrated in granule cells than Purkinje cells, whereas the opposite appears true for G_Tla. It also appears that GD3 is enriched in reactive glial cells and may play an important role during the morphological transformation of neural membranes.     

Identification of genes differentially expressed in wild type and Purkinje cell degeneration mice

Purkinje cell degeneration (pcd) mice are characterized by death of virtually all cerebellar Purkinje cells by postnatal day 30. In this study, we used DNA microarray analysis to investigate differences in gene expression between the brains of wild type and pcd mice on postnatal day 20, before the appearance of clear-cut phenotypic abnormalities. We identified 300 differentially expressed genes, most of which were involved in metabolic and physiological processes. Among the differentially expressed genes were several calcium binding proteins including calbindin-28k, paravalbumin, matrix gamma-carboxyglutamate protein and synaptotagamins 1 and 13, suggesting the involvement of abnormal Ca2+ signaling in the pcd phenotype.     

Nna1 mediates Purkinje cell dendritic development via lysyl oxidase propeptide and NF-κB signaling

The molecular pathways controlling cerebellar Purkinje cell dendrite formation and maturation are poorly understood. The Purkinje cell degeneration (pcd) mutant mouse is characterized by mutations in Nna1, a gene discovered in an axonal regenerative context, but whose actual function in development and disease is unknown. We found abnormal development of Purkinje cell dendrites in postnatal pcd(Sid) mice and linked this deficit to a deletion mutation in exon 7 of Nna1. With single cell gene profiling and virus-based gene transfer, we analyzed a molecular pathway downstream to Nna1 underlying abnormal Purkinje cell dendritogenesis in pcd(Sid) mice. We discovered that mutant Nna1 dramatically increases intranuclear localization of lysyl oxidase propeptide, which interferes with NF-κB RelA signaling and microtubule-associated protein regulation of microtubule stability, leading to underdevelopment of Purkinje cell dendrites. These findings provide insight into Nna1's role in neuronal development and why its absence renders Purkinje cells more vulnerable.     

A cerebellar Purkinje cell marker P400 protein is an inositol 1,4,5-trisphosphate (InsP3) receptor protein. Purification and characterization of InsP3 receptor complex.

P400 protein is a 250 kd glycoprotein, characteristic of the cerebellum, which is accumulated at the endoplasmic reticulum, at the plasma membrane and at the post-synaptic density of Purkinje cells. In this study, we purified inositol 1,4,5-trisphosphate (InsP3) receptor from mouse cerebellum and examined the possibility that P400 protein is identical with cerebellar InsP3 receptor protein. InsP3 receptor was solubilized with Triton X-100 from a post-nuclear fraction of ddY mouse cerebellum and was purified with high yield by sequential column chromatography on DE52, heparin-agarose, lentil lectin-Sepharose and hydroxylapatite. In these chromatographies, P400 protein co-migrated completely with the InsP3 binding activity. The purified receptor is a 250 kd protein with a Bmax of 2.1 pmol/microgram and a KD of 83 nM. It reacted with three different monoclonal antibodies against P400 protein, indicating that P400 protein is the same substance as the InsP3 receptor (P400/InsP3 receptor protein). Electron microscopy of the purified receptor showed a square shape with sides approximately 25 nm long. Binding assays of the cerebella of Purkinje cell-degeneration (pcd) mice with [3H]InsP3 demonstrated that the InsP3 binding sites in the cerebellum are distributed exclusively on the Purkinje cells. Immunohistochemical analysis indicated that P400/InsP3 receptor is present at the dendrites, cell bodies, axons and synaptic boutons of the Purkinje cells.     

Cultivation of Purified Primary Purkinje Cells from Rat Cerebella

Primary neurons are difficult to cultivate because they are often part of a complex tissue, and synaptically connected to numerous other cell types. These circumstances often prevent us from unveiling molecular and metabolic mechanisms of distinct cells, as functional signals or assays cannot clearly be correlated with them due to interfering signals from other parts of the culture. We therefore present an up-to-date method for obtaining a highly purified neuronal culture of Purkinje cells. In the past, Purkinje cells were successfully isolated from young mouse cerebella, but this protocol was never adapted to other mammals. We therefore provide an updated and adjusted protocol for Purkinje cell isolation from rat instead of mouse cerebella. To purify Purkinje cells, we obtained perinatal rat cerebella, dissociated them and performed a Percoll gradient centrifugation to segregate the smaller and larger cell fractions. In a second step, we performed an immunopanning procedure to enrich only Purkinje cells from the large cell fraction. Based on former protocols, we used a different antibody for the immunopanning procedure and adjusted several aspects from the initial protocol to improve the yield and vitality of Purkinje cells. We provide RT-qPCR-based purity data obtained with this protocol and show the behaviour and the growth of these purified Purkinje cells. We provide a highly reproducible purification protocol for Purkinje cell cultures of high purity that allows functional analysis and downstream assays on living rat Purkinje cells and further morphological growth analysis in future.     

Presence of protein I, a phosphoprotein associated with synaptic vesicles, in cerebellar granule cells

Abstract: The cerebellar levels of Protein I, a synapse-specific neuronal phosphoprotein, have been investigated in the cerebellar mouse mutants staggerer ( sg ), weaver ( wv ), nervous ( nr ), and Purkinje cell degeneration ( pcd ). The Protein I concentration was reduced by about 66% in sg and wv mutants, representing a 90% loss of Protein I per cerebellum. A heterozygote effect was observed in the wv mutant. These results indicate that a great majority of Protein I in the normal cerebellum may be present in the granule cells. in nr mutants the cerebellar Protein I concentration was reduced by only 12% in 62-day-old mice, suggesting that Purkinje cells contribute little to cerebellar Protein I. However, a greater reduction was observed in pcd mutants, which may reflect on the nature of the pcd mutation.     

Analysis of transcriptional profiles and functional clustering of global cerebellar gene expression in PCD3J mice

The Purkinje cell degeneration (PCD) mutant mouse is characterized by a degeneration of cerebellar Purkinje cells and progressive ataxia. To identify the molecular mechanisms that lead to the death of Purkinje neurons in PCD mice, we used Affymetrix microarray technology to compare cerebellar gene expression profiles in pcd3J mutant mice 14 days of age (prior to Purkinje cell loss) to unaffected littermates. Microarray analysis, Ingenuity Pathway Analysis (IPA) and expression analysis systematic explorer (EASE) software were used to identify biological and molecular pathways implicated in the progression of Purkinje cell degeneration. IPA analysis indicated that mutant pcd3J mice showed dysregulation of specific processes that may lead to Purkinje cell death, including several molecules known to control neuronal apoptosis such as Bad, CDK5 and PTEN. These findings demonstrate the usefulness of these powerful microarray analysis tools and have important implications for understanding the mechanisms of selective neuronal death and for developing therapeutic strategies to treat neurodegenerative disorders.     

Neuronal influence on glial enzyme expression: evidence from chimeric mouse cerebellum

Cerebella, variably deficient in Purkinje cells, were obtained from aggregation chimeras of either Lurcher or Purkinje cell degeneration mutants. These cerebella were used to analyze the expression of glycerol-3-phosphate dehydrogenase (GPDH) in Bergmann glia. Immunocytochemistry showed apparently normal GPDH expression only in Bergmann glia in the immediate vicinity of surviving Purkinje cells. The number of GPDH-positive Bergmann glia cells associated with isolated Purkinje cells was close to that expected, based on measurements in Golgi-stained, normal cerebella of the Bergmann glia cell's domain. The results support the hypothesis that GPDH expression in Bergmann glia cells depends upon their sustained interaction with Purkinje cells, most likely involving direct cell-cell contact.     

Abnormal sperm development in pcd 3J-/- mice: the importance of Agtpbp1 in spermatogenesis

Homozygous Purkinje cell degeneration (pcd) mutant males exhibit abnormal sperm development. Microscopic examination of the testes from pcd(3J)-/- mice at postnatal days 12, 15, 18 and 60 revealed histological differences, in comparison to wild-type mice, which were evident by day 18. Greatly reduced numbers of spermatocytes and spermatids were found in the adult testes, and apoptotic cells were identified among the differentiating germ cells after day 15. Our immunohistological analysis using an antihuman AGTPBP1 antibody showed that AGTPBP1 was expressed in spermatogenic cells between late stage primary spermatocytes and round spermatids. A global gene expression analysis from the testes of pcd(3J)-/- mice showed that expression of cyclin B3 and de-ubiquitinating enzymes USP2 and USP9y was altered by >1.5-fold compared to the expression levels in the wild-type. Our results suggest that the pcd mutant mice have defects in spermatogenesis that begin with the pachytene spermatocyte stage and continue through subsequent stages. Thus, Agtpbp1, the gene responsible for the pcd phenotype, plays an important role in spermatogenesis and is important for survival of germ cells at spermatocytes stage onward.     

Dysmyelination of Shiverer Mutant Mice In Vivo and In Vitro

Demyelination in the CNS of shiverer mutant mice was studied in vivo and in vitro. By immunohistochemical reaction with glial fibrillary acidic protein antibody, hypertrophy of the fibrous astrocytes was observed in the white matter of shiverer cerebella. The cerebella of shiverer mice in primary culture from the day of birth showed very poor myelination under optical microscopy. Axons of Purkinje cells are thought to be the main myelinated axons in the primary culture of the cerebellum. Purkinje cells from shiverer appeared normal with regard to Bodian silver impregnation, hematoxylin and eosin staining, and P400 protein characterization of Purkinje cells. Addition of the conditioned culture medium of shiverer to the control culture did not interfere with myelination. We concluded that the demyelination in the CNS of shiverer could be caused by an intrinsic defect of the oligodendrocyte rather than by hypertrophy of the astrocytes or by diffusible factors.     

Two-Dimensional Gel Electrophoretic Analysis of the Cerebella from Neuro-Pathologically-Mutant Weaver,Nervous and Staggerer Mice1

We have analysed the proteins of the cerebella from mutant and control mice by applying high resolution two-dimensional polyacrylamide gel electrophoresis. The tissue of each cerebellum and also the pallium cerebri were fractionated into water-soluble and particulate fractions, and these were used in gel electrophoresis. In order to augment the sensitivity for detection of protein spots, we applied silver staining. We used the cerebella from weaver (granule cell deficient), nervous (Purkinje cell deficient), and staggerer (poor dendritic arborization of Purkinje cells) mutant mice. The present technique revealed at least 700 to 800 protein spots. Among the spots detected we found 12 new significantly-changed proteins in the cerebella of the mutants. The possible significance of these proteins is discussed.     

Growth arrest specific gene 1 is a positive growth regulator for the cerebellum.

Postnatal cerebellum development involves the generation of granule cells and Bergmann glias (BGs). The granule cell precursors are located in the external germinal layer (EGL) and the BG precursors are located in the Purkinje layer (PL). BGs extend their glial fibers into the EGL and facilitate granule cells' inward migration to their final location. Growth arrest specific gene 1 (Gas1) has been implicated in inhibiting cell-cycle progression in cell culture studies (G. Del Sal et al., 1992, Cell 70, 595--607). However, its growth regulatory function in the CNS has not been described. To investigate its role in cerebellar growth, we analyzed the Gas1 mutant mice. At birth, wild-type and mutant mice have cerebella of similar size; however, mature mutant cerebella are less than half the size of wild-type cerebella. Molecular and cellular examinations indicate that Gas1 mutant cerebella have a reduced number of granule cells and BG fibers. We provide direct evidence that Gas1 is required for normal levels of proliferation in the EGL and the PL, but not for their differentiation. Furthermore, we show that Gas1 is specifically and coordinately expressed in both the EGL and the BGs postnatally. These results support Gas1 as a common genetic component in coordinating EGL cell and BG cell proliferation, a link which has not been previously appreciated.     

Cytosolic carboxypeptidase 1 is involved in processing α- and β-tubulin

The Purkinje cell degeneration (pcd) mouse has a disruption in the gene encoding cytosolic carboxypeptidase 1 (CCP1). This study tested two proposed functions of CCP1: degradation of intracellular peptides and processing of tubulin. Overexpression (2-3-fold) or knockdown (80-90%) of CCP1 in human embryonic kidney 293T cells (HEK293T) did not affect the levels of most intracellular peptides but altered the levels of α-tubulin lacking two C-terminal amino acids (delta2-tubulin) ≥ 5-fold, suggesting that tubulin processing is the primary function of CCP1, not peptide degradation. Purified CCP1 produced delta2-tubulin from purified porcine brain α-tubulin or polymerized HEK293T microtubules. In addition, CCP1 removed Glu residues from the polyglutamyl side chains of porcine brain α- and β-tubulin and also generated a form of α-tubulin with two C-terminal Glu residues removed (delta3-tubulin). Consistent with this, pcd mouse brain showed hyperglutamylation of both α- and β-tubulin. The hyperglutamylation of α- and β-tubulin and subsequent death of Purkinje cells in pcd mice was counteracted by the knock-out of the gene encoding tubulin tyrosine ligase-like-1, indicating that this enzyme hyperglutamylates α- and β-tubulin. Taken together, these results demonstrate a role for CCP1 in the processing of Glu residues from β- as well as α-tubulin in vitro and in vivo.     

Cyclic Nucleotide-Dependent Protein Kinases and Some Major Substrates in the Rat Cerebellum After Neonatal X-Irradiation

The levels of cAMP-dependent protein kinase (type I), or cGMP-dependent protein kinase, or protein I, and of a 23,000 MW substrate for the cGMP-dependent protein kinase were measured in cerebella from normal rats and in the cerebella from rats in which a selective loss of interneurons in the cerebellar cortex had been produced by X-irradiation. A decrease was observed in the concentrations of cAMP-dependent protein kinase and of protein I, whereas an increase was observed in the concentrations of cGMP-dependent protein kinase and of the 23,000 MW substrate. The data, taken together with the results of other studies, support the interpretation that cAMP-dependent protein kinase and protein I are distributed throughout the cerebellum, but that cGMP-dependent protein kinase and the 23,000 MW substrate are highly concentrated in Purkinje cells.     

Abnormal Purkinje cell dendrites in Lurcher chimeric mice result from a deafferentation-induced atrophy

Previous studies of Purkinje cell dendrites in lurcher ↔ wild-type mouse chimeras (lurcher chimeras) have documented the surprising occurrence of unusual atrophic dendritic morphologies among the wild-type cells of the mosaic cerebella. We have hypothesized that these aberrant morphologies arise from a process of developmental deafferentation that is due to the unique loss of mutant Purkinje cells in these chimeras. These earlier studies left unanswered the question of whether the abnormal dendrites were the result of a blocked developmental process (agenesis) or regressive events that deform a previously well-developed dendritic arbor (atrophy). Using a set of simple morphometric measures, we now examine wild-type Purkinje cells in young lurcher chimeras. At postnatal day 20, normal Purkinje cell development is nearly but not fully complete. In lurcher chimeras, the morphologies of the wild-type Purkinje cell dendrites are similar to those in wild-type controls of the same age. This means that they are larger in height, width, and cross-section than their counterparts in adult lurcher chimeras. The younger cells exhibit almost none of the atrophic morphologies described in mature animals. We conclude that the aberrant morphologies found in adult lurcher chimeras arise from atrophy rather than through a failure in development. Furthermore, consideration of the details of the wild-type dendrites in the lurcher chimeras leads to the proposal that the height and width of the Purkinje cell dendritic tree are controlled by two independent mechanisms. © 1996 John Wiley & Sons, Inc.     

Effect of nonionizing radiation on the purkinje cells of the uvula in squirrel monkey cerebellum

Pregnant squirrel monkeys were exposed to 2450-MHz (CW) microwaves at an equivalent power density of 10 mW/cm² (SAR 3.4 mW/g) for three hours daily in a cavity-cage module. The exposure began when pregnancy was determined by a hormonal method, and continued through the offspring's first 9.5 months. After irradiation, the brains of the offspring were fixed with formaldehyde, and the inferior vermis of each cerebella was removed and processed for histologic observations. Purkinje cell density in the uvula was determined in sagittal serial sections. There was no significant difference between control and experimental animals in the number of Purkinje cells per mm of Purkinje cell line (linear density), as well as in the density of Purkinje cells in the Purkinje cell layer.