Preventive motor training but not progenitor grafting ameliorates cerebellar ataxia and deregulated autophagy in tambaleante mice.

Treatment options for degenerative cerebellar ataxias are currently very limited. A large fraction of such disorders is represented by hereditary cerebellar ataxias, whose familiar transmission facilitates an early diagnosis and may possibly allow to start preventive treatments before the onset of the neurodegeneration and appearance of first symptoms. In spite of the heterogeneous aetiology, histological alterations of ataxias often include the primary degeneration of the cerebellar cortex caused by Purkinje cells (PCs) loss. Thus, approaches aimed at replacing or preserving PCs could represent promising ways of disease management. In the present study, we compared the efficacy of two different preventive strategies, namely cell replacement and motor training. We used tambaleante (tbl) mice as a model for progressive ataxia caused by selective loss of PCs and evaluated the effectiveness of the preventive transplantation of healthy PCs into early postnatal tbl cerebella, in terms of PC replacement and functional preservation. On the other hand, we investigated the effects of motor training on PC survival, cerebellar circuitry and their behavioral correlates. Our results demonstrate that, despite a good survival rate and integration of grafted PCs, the adopted grafting protocol could not alleviate the ataxic symptoms in tbl mice. Conversely, preventive motor training increases PCs survival with a moderate positive impact on the motor phenotype.

Binuclear Purkinje neurons.

Until the end of the XX century binuclear neurons of Purkinje in rodents and the humans were a subject of casual finds. However already then it was noticed that such cells are in old and sick mammals more often. It is therefore assumed that the appearance of the second nucleus has a regenerative value - compensation age-related or pathogenic loss of Purkinje cells. In 2003, in research on stem cell transplantation was made the first observations related to the mechanism of the appearance of the second nucleus in Purkinje neurons. The transgender studies in humans and in transgenic experiments on mice have shown that bone marrow derived donor cells can fuse with Purkinje neurons of the recipient, thus transfer to neuron its nucleus. It is very important that the binuclear neurons can appear in old and sick people and rodents without transplantation. But in that case neither the donor cell, nor the mechanism of origin of the second nucleus remain not clear. Relevance of clarification of this question increases of the fact that literature of the last years proves: emergence of the second nucleus is a form of physiological and reparative regeneration of neurons of Purkinje.

Differentiation of human induced pluripotent stem cells to mature functional Purkinje neurons.

It remains a challenge to differentiate human induced pluripotent stem cells (iPSCs) or embryonic stem (ES) cells to Purkinje cells. In this study, we derived iPSCs from human fibroblasts and directed the specification of iPSCs first to Purkinje progenitors, by adding Fgf2 and insulin to the embryoid bodies (EBs) in a time-sensitive manner, which activates the endogenous production of Wnt1 and Fgf8 from EBs that further patterned the cells towards a midbrain-hindbrain-boundary tissue identity. Neph3-positive human Purkinje progenitors were sorted out by using flow cytometry and cultured either alone or with granule cell precursors, in a 2-dimensional or 3-dimensional environment. However, Purkinje progenitors failed to mature further under above conditions. By co-culturing human Purkinje progenitors with rat cerebellar slices, we observed mature Purkinje-like cells with right morphology and marker expression patterns, which yet showed no appropriate membrane properties. Co-culture with human fetal cerebellar slices drove the progenitors to not only morphologically correct but also electrophysiologically functional Purkinje neurons. Neph3-posotive human cells could also survive transplantation into the cerebellum of newborn immunodeficient mice and differentiate to L7- and Calbindin-positive neurons. Obtaining mature human Purkinje cells in vitro has significant implications in studying the mechanisms of spinocerebellar ataxias and other cerebellar diseases.

Selective rather than inductive mechanisms favour specific replacement of Purkinje cells by embryonic cerebellar cells transplanted to the cerebellum of adult Purkinje cell degeneration (pcd) mutant mice.

Cell replacement after neuronal degeneration in the adult CNS depends on the availability of specific cues to direct specification, differentiation and integration of newly born neurons into mature circuits. Following recent reports indicating that neurogenic signals may be reactivated in the adult injured CNS, here we asked whether such signals are expressed in the cerebellum after Purkinje cell degeneration. Thus, we compared the fate of embryonic cerebellar cells transplanted to the cerebella of adult wild-type and Purkinje cell degeneration (pcd) mutant mice. Donor cells were dissected from beta-actin-enhanced green fluorescent protein (EGFP) transgenic mice and transplanted as a single cell suspension. In both hosts, grafted cells generated all major cerebellar phenotypes, with a precise localization in the recipient cortex or white matter. Nevertheless, the phenotypic distributions showed striking quantitative differences. Most notably, in the pcd cerebellum there was a higher amount of Purkinje cells, while other phenotypes were less frequent. Analysis of cell proliferation by 5-bromo-2'-deoxyuridine (BrDU) incorporation revealed that in both hosts mitotic activity was strongly reduced shortly after transplantation, and virtually all donor Purkinje cells were actually generated before grafting. Together, these results indicate that some compensatory mechanisms operate in the pcd environment. However, the very low mitotic rate of transplanted cells suggests that the adult cerebellum, either wild-type or mutant, does not provide instructive neurogenic cues to direct the specification of uncommitted progenitors. Rather, specific replacement in mutant hosts is achieved through selective mechanisms that favour the survival and integration of donor Purkinje cells at the expense of other phenotypes.

Various methods of Purkinje cells transplantation and their functional response in Lurcher mutant mice.

Embryonic cerebellum was transplanted to adult Lurcher mutant mice affected with hereditary olivocerebellar degeneration and with resulting cerebellar ataxia. Grafts were applied as solid pieces of tissue or as cell suspensions. The aim was to replace Purkinje cells lost by the neurodegeneration with embryonic cells and to observe the effect on motor symptoms of cerebellar ataxia. Success rate of the two methods was also compared. Motor skills were tested before and in week intervals after the transplantation. The results were compared with sham-operated controls. When the solid graft was transplanted, the success rate was two times higher as compared with the cell suspension method. Fibre sprouting and cell migration from the graft to the host tissue was observed. Insignificant amelioration of motor skills was found in mice after the solid cerebellar tissue transplantation, while the cell suspension application had no effect.

Cell-autonomous mechanisms and myelin-associated factors contribute to the development of Purkinje axon intracortical plexus in the rat cerebellum.

The highly specific connection patterns of the mature CNS are shaped through finely regulated processes of axon growth and retraction. To investigate the relative contribution of cell-autonomous mechanisms and extrinsic cues in these events, we examined the development of Purkinje axon intracortical plexus in the rat cerebellum. During the first postnatal week, several new processes sprout from focal swellings along the initial portion of the Purkinje neurite and spread in the granular layer. Intense structural plasticity occurs during the following week, with pruning of collateral branches and remodeling of terminal arbors. The mature distribution of the Purkinje infraganglionic plexus, confined within the most superficial portion of the granular layer, is attained at approximately postnatal day 15. A similar neuritic branching pattern is also developed by Purkinje cells grown in cultures of dissociated cerebellar cells or transplanted to extracerebellar CNS regions, suggesting that cell-autonomous mechanisms contribute to determining the Purkinje axon phenotype. The structural remodeling of Purkinje intracortical plexus is concomitant with the development of cerebellar myelin. To ask whether myelin-associated factors contribute to the morphological maturation of Purkinje neurites, we prevented normal myelinogenesis by killing oligodendrocyte precursors with 5'-azacytidine or by applying neutralizing antibodies against the myelin-associated neurite growth inhibitor Nogo-A. In both conditions, Purkinje axons retained exuberant branches, and the terminal plexus spanned the entire extent of the granular layer. Thus, the formation of Purkinje axon collaterals is, in part, controlled by intrinsic determinants, but their growth and distribution are regulated by environmental signals, among which are myelin-derived cues.

Purkinje cell transplants in Shaker mutant rats with hereditary Purkinje cell degeneration and ataxia.

Shaker mutant rats are characterized by the adult-onset degeneration of cerebellar anterior lobe Purkinje cells and temporally correlated development of ataxia and tremor. Normal E-13 Purkinje cells were transplanted into the anterior cerebellum in adult shaker mutant rats to study donor/host interactions in an animal with adult-onset heredodegeneration. Donor Purkinje cells from extraparenchymal transplant sites migrated radially into the host molecular layer and differentiated. Donor Purkinje cell dendrites expanded to fill the host molecular layer, spinous processes were apparent, and axonal projections into the host gray and white matter were observed. Donor Purkinje cells remaining in the extraparenchymal transplant sites differentiated if they were located relatively close to the host cerebellum. Donor Purkinje cells located intraparenchymally in the host white matter or granule cell layer survived, but were stunted in their development. The orthogonal movement of donor Purkinje cells away from transplant sites in the host cerebellum was spatially restricted. The findings from this study indicate that host cerebellar cortex with adult-onset heredodegeneration of Purkinje cells supports the survival and differentiation of transplanted normal embryonic Purkinje cells.

Transplanted neurons alter the course of neurodegenerative disease in Lurcher mutant mice.

Embryonic cerebellar, neocortical, and striatal tissues derived from NSE-LacZ transgenic mice were transplanted into the right cerebellar hemisphere of 8- to 10-day-old Lurcher or wild-type mice. Host mice survived for 30-90 days and the transplanted tissue was examined by light microscopy using Nissl staining, X-gal histochemistry, and immunohistochemistry for calcium binding protein and glutamic acid decarboxylase. Transplantation of cerebellar tissue, but not neocortical or striatal progenitors, resulted in robust infiltration of the lurcher mutant host cerebellar cortex by transgenic Purkinje neurons. Deep to the infiltrated molecular layer, the host granular layer was thicker and denser than the mutant granular layer, but transgenic cells did not contribute to the spared granular layer. The host inferior olivary complex consistently exhibited a noticeable bilateral asymmetry in Nissl-stained sections. A quantitative analysis of the olivary complex was performed in 10 90-day-old host mice. The results indicate that the left inferior olivary complex of 90-day-old host mice contained more neurons than the right inferior olive of the host mice and contained more neurons than was observed in 90-day-old Lurcher control mice. Analysis by olivary subdivision indicates that increased neuron numbers were present in all subdivisions of the host left inferior olive. These studies confirm the specific attractive effect of the mutant cerebellar cortex on transplanted Purkinje neuron progenitors and indicate that neural transplants may survive the neurodegenerative period to interact with developing host neural systems. The unilateral rescue of Lurcher inferior olivary neurons in cerebellar transplant hosts indicates that transplanted neurons may interact with diseased host neural circuits to reduce transneuronal degeneration in the course of a neurodegenerative disease.

Cerebellar grafts partially reverse amino acid receptor changes observed in the cerebellum of mice with hereditary ataxia: quantitative autoradiographic studies.

We used quantitative autoradiography of [3H]CNQX (200 nM), [3H]muscimol (13 nM), and [3H]flunitrazepam (10 nM) binding to study the distribution of non-NMDA and GABA(A) receptors in the cerebellum of pcd mutant mice with unilateral cerebellar grafts. Nonspecific binding was determined by incubation with 1 mM Glu, 200 microM GABA, or 1 microM clonazepam, respectively. Saturation parameters were defined in wild-type and mutant cerebella. In mutants, non-NMDA receptors were reduced by 38% in the molecular layer and by 47% in the granule cell layer. The reduction of non-NMDA receptors in the pcd cerebellar cortex supports their localization on Purkinje cells. [3H]CNQX binding sites were visualized at higher density in grafts that had migrated to the cerebellar cortex of the hosts (4.1 and 11.0 pmol/mg protein, respectively, at 23 and 37 days after grafting) than in grafts arrested intraparenchymally (2.6 and 6.2 pmol/mg protein, respectively, at 23 and 37 days after grafting). The pattern of expression of non-NMDA receptors in cortical vs. parenchymal grafts suggests a possible regulation of their levels by transacting elements from host parallel fibers. GABA(A) binding levels in the grafts for both ligands used were similar to normal molecular layer. Binding was increased in the deep cerebellar nuclei of pcd mutants: the increase in [3H]muscimol binding over normal was 215% and the increase in [3H]flunitrazepam binding was 89%. Such increases in the pcd deep cerebellar nuclei may reflect a denervation-induced supersensitivity subsequent to the loss of Purkinje axon terminal innervation. In the deep nuclei of pcd mutants with unilateral cerebellar grafts, [3H]muscimol binding was 31% lower in the grafted side than in the contralateral nongrafted side at 37 days after transplantation; [3H]flunitrazepam binding was also lower in the grafted side by 15% compared to the nongrafted side. Such changes in GABA(A) receptors suggest a significant, albeit partial, normalizing trend of cerebellar grafts on the state of postsynaptic supersensitive receptors in the host cerebellar nuclei.

Regulation of Purkinje cell alignment by reelin as revealed with CR-50 antibody.

Cerebellar Purkinje cells are generated in the ventricular zone, migrate outward, and finally form a monolayer in the cortex. In reeler mice, however, most Purkinje cells cluster abnormally in subcortical areas. Reelin, the candidate reeler gene product recognized by the CR-50 monoclonal antibody, is concentrated in a cortical zone along which Purkinje cells are aligned linearly, implying that it may regulate their alignment. We used an in vitro system and a transplantation approach to analyze the function of Reelin. Explant culture for 7 d of cerebella isolated from wild-type and reeler mice at embryonic day 13 (E13) reproduced in a phenotype-dependent manner the two distinct arrangement patterns (linear vs clustered) of Purkinje cells. Extensive CR-50 binding to wild-type explants converted the linear pattern into a reeler-like, clustered pattern. On the other hand, when reeler explants lacking Reelin were crowned with an artificial layer of Reelin+ granule cells, some Reelin molecules were distributed into a superficial zone of the reeler explants, and Purkinje cells formed a linear pattern along the Reelin-rich overlay. This "rescue" effect was also inhibited by CR-50. Hence, Reelin is involved in the Purkinje cell alignment, and the lack of this activity may explain the malformation in reeler cerebella. We further injected Reelin+ granule cells into the fourth ventricle of E12-13 mice. Extensive incorporation of the injected Reelin+ cells into the ventricular zone, but not of Reelin- cells, forced Purkinje cells of the host cerebella to form an aberrant layer, suggesting that premigratory Purkinje cells may already be responsive to Reelin or Reelin-related signals.

Postsynaptic currents and short-term synaptic plasticity in Purkinje cells grafted onto an uninjured adult cerebellar cortex.

It has been shown recently that embryonic Purkinje cells grafted extraparenchymally into an intact cerebellum, in the absence of any sign of damage, are able to migrate into the host molecular layer where they receive a climbing fibre innervation. Using the same technique, we investigated the development of the electrophysiological properties of the synapses between the grafted cells and their main afferents. Purkinje cells either in the graft or having migrated into the molecular layer of the host were recorded using the whole-cell patch-clamp method in acutely prepared slices 17-112 days after grafting. Spontaneous postsynaptic currents with a single-exponential decay and mediated by GABAA receptors were very similar to those described in normal Purkinje cells. Excitatory postsynaptic currents (EPSCs) evoked by climbing fibre and by parallel fibre stimulation were blocked by an alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA)/kainate antagonist, and displayed the linear current-voltage relation typical of postnatal Purkinje cells. The attainment of normal functional properties by the adult axons at the newly formed synaptic sites was shown by the expression of short-term facilitation of parallel fibre EPSCs and of short-term depression of climbing fibre EPSCs. The grafted Purkinje cells showed climbing fibre polyinnervation 17-20 days after grafting which evolved to monoinnervation at 23-45 days, confirming the completion of the developmental programme up to maturation. Our experiments support the view that the adult intact brain is able to accept and integrate an additional number of neurons which show fully mature electrophysiological properties which are electrophysiologically indistinguishable from those of the host neurons.

Amelioration of the behavioral phenotype in genetically ataxic mice through bilateral intracerebellar grafting of fetal Purkinje cells.

We have previously applied neural grafting to "Purkinje cell degeneration" mutant mice (gene symbol pcd, mouse chromosome 13), a model of recessively inherited cerebello-olivary atrophy, to create appropriate interactions between wild-type and mutant cells in elucidating gene effects on the involved neuron populations and to address issues of the structural integration of donor Purkinje cells into the disrupted cerebellar loop. Behaviorally, pcd homozygotes manifest ataxic signs beginning at 3-4 wk of age. The functional effects of cerebellar transplants on motor performance have long remained an open question. The aim of the present study was to determine the recovery of motor responses in pcd mutants in a battery of behavioral tasks after bilateral transplantation of cerebellar cell suspensions (prepared from wild-type mice) into the parenchyma of the deep cerebellar nuclei of the hosts, according to a protocol that emphasizes the reconstruction of the missing inhibitory cortico-nuclear projection. With this approach, the denervated deep nuclei of the host receive a new Purkinje axonal innervation; further, most transplanted Purkinje cells end up occupying cortical localities anyway and display a correct dendritic tree orientation toward the pia. Motor coordination and fatigue resistance were assessed in a rotarod treadmill apparatus, a behavioral paradigm useful in studying various brain abiotrophies and treatments, including developmental perturbations of the cerebellar cytoarchitecture. Locomotor activity was quantified by the number of squares mice crossed as they moved about in an open-field matrix. Grafted pcd mice performed significantly better than sham-operated mutants in both of these tasks. Moreover, graft-recipient mice were able to sustain their abdomen above the floor on their limbs during movement, contrasting to the typical lowered, widened stance of sham-operated pcd mutants. These findings clearly demonstrate that bilateral transplants of fetal Purkinje cells have functional effects on motor performance in the pcd model of hereditary cerebellar ataxia.

Molecular plasticity of adult Bergmann fibers is associated with radial migration of grafted Purkinje cells.

Embryonic Purkinje cells (PCs) from cerebellar primordia grafted in adult pcd mutant cerebellum replace missing PCs of the host, and become synaptically integrated into the defective cerebellar circuit. This process of neuronal replacement starts with the invasion of grafted PCs into the host cerebellum, and their radial migration through its molecular layer. The present study is aimed at determining whether the glial axes for this migration are embryonic radial glial cells that comigrate with the grafted PCs, or adult Bergmann fibers of the host, transiently reexpressing the molecular cues needed for their guidance of the migration. Transplants from a transgenic mouse line (Krox-20/lacZ14) in which Bergmann fibers could be identified by lacZ expression reveal that, despite the presence of X-gal-stained Bergmann fibers in the graft remnants and of grafted PCs in the host molecular layer, all Bergmann fibers in the host cerebellum lack of beta-galactosidase activity. Thus, these migratory axes belong to the host, not to the donor. Transplants from normal isogenic mouse embryos show that during the radial migration of grafted PCs (7 d after grafting) the involved host Bergmann fibers reexpress nestin (identified with monoclonal antibody Rat-401 immunostaining), normally expressed only by immature Bergmann fibers. Five days later, when grafted PCs have arrested their migration, host Bergmann fibers again become Rat-401 negative. These results indicate that embryonic PCs can trigger in adult cerebellum the molecular changes necessary for their own migration and ultimate synaptic integration in the host cortical circuitry.

The dorsal cochlear nucleus of the adult lurcher mouse is specifically invaded by embryonic grafted Purkinje cells.

The fate of embryonic Purkinje cells grafted over the brainstem surface of the adult Lurcher mouse was analyzed using anti-calbindin (CaBP) immunocytochemistry. Purkinje cells are able to migrate specifically into the molecular layer of the host dorsal cochlear nucleus (DCoN) and develop dendritic trees that are practically isoplanar, suggesting synaptic interactions with the parallel fibres of the DCoN. These results provide a new argument in favour of the homology between the cerebellum and the DCoN.

Bergmann glia require continuous association with Purkinje cells for normal phenotype expression.

Bergmann glia (Bg) respond to the early postnatal Purkinje cell (Pc) death in Lurcher (Lc) mutant mouse cerebellum by down-regulating expression of the enzyme glycerol-3-phosphate dehydrogenase (GPDH). To determine whether glial GPDH expression requires the continued presence of Pcs in adults, we used single intracerebellar injections of kainic acid to kill Pcs in wild-type mice from 7 weeks to 11 months old. Bg at all ages tested responded to Pc loss by down-regulating GPDH expression. To learn whether a high level of GPDH could be reinduced following down-regulation in Lc Bg, we grafted wild-type fetal Pcs into Lc cerebella. The influence of grafted Pcs on GPDH expression is host-age and implant-position dependent. Only Pcs implanted into hosts less than 6 weeks old were later found to be associated with GPDH-positive Bg. Grafted Pcs that migrated into the anterior folia of young hosts were more likely to be associated with GPDH-positive Bg than Pcs migrating to other positions. EM analysis showed that Bg ensheathment of grafted Pcs is thinner and more discontinuous, but qualitatively similar to normal. The results suggest that the interaction between host Bg and grafted Pcs can sustain elevated GPDH expression in Bg that have not yet down-regulated, but is not adequate to reinduce expression in those cells that have.

Transplantation of normal embryonic cerebellar cell suspensions into the cerebellum of lurcher mutant mice.

Injection of cell suspensions of normal embryonic cerebellar tissue into adult and juvenile lurcher mutant mice results in infiltration of the atrophic host cerebellar cortex by genetically normal Purkinje cells, as has previously been observed in Purkinje cell degeneration (pcd) mutant mice. In lurcher, the grafted Purkinje neurons most frequently occupy the host molecular layer and produce axons which, in some cases, invade the host cerebellar nuclei. The grafted Purkinje neurons fail to adopt their characteristic planar dendritic disposition in lurcher hosts, probably because of the severe depletion of cerebellar granule neurons and parallel fibers in this mutant. Results reported previously in pcd mutants have thus been extended, with some deviations, to a similar but genetically distinct hereditary cerebellar atrophy, providing evidence of the generalized nature of the phenomena involved.

Mutant mouse cerebellum does not provide specific signals for the selective migration and development of transplanted Purkinje cells.

Embryonic cerebellum transplanted to adult Purkinje cell degenerate mice was assessed for integration and Purkinje cell migration by using the antigenic markers Thy-1 and Leu-4. It was found that the grafted cells migrated into the host's molecular layer, but there was no evidence for specific migration of Purkinje cells. Furthermore, grafted cells were found to form normal cerebellar cyto-architecture only with other grafted cells and not with the host's cells.

[Migration of donor Purkinje cells in the host adult rat cerebellum].

It is considered that cell adhesion molecules play important roles in the host-graft interaction during the reconstruction of the injured nervous system by neural transplantation. In this article, we report the expression of such molecules during the migration and differentiation of donor Purkinje cells in the adult rat cerebellum. Cerebellar primordium at the 14th day of gestation was transplanted into the adult rat cerebellum. Purkinje cells which had migrated from the grafted tissue into the host molecular layer were identified immunohistochemically with anti-spot 35 antibody, a specific marker for Purkinje cells in the cerebellum, as well as by labeling them with bromodeoxyuridine at their final mitotic period. In the grafted site, transient expression of a neuron-glia cell adhesion molecule, tenascin, was detected immunohistochemically. This molecule was expressed in the host tissue adjacent to the migratory Purkinje cells as well as within the donor immature tissue. Tenascin was not detected in the host tissue far distant from the grafted tissue. In considering the expression of tenascin in the migratory process of Purkinje cells during cerebellar development, this molecule induced in the host tissue may be involved in the migration of donor Purkinje cells.

Serotonin fiber innervation of cerebellar cell suspensions intraparenchymally grafted to the cerebellum of pcd mutant mice.

One aspect of integration of implanted neurons into the neuronal circuitry of a defective host brain is the re-establishment of a host-to-graft afferent innervation. We addressed this issue by using the adult cerebellum of 'Purkinje cell degeneration' (pcd) mutant mice, which lack virtually all Purkinje cells after postnatal day (P) 45. Purkinje cells constitute one of the cerebellar cell types being innervated by axons of raphé serotonin (5-HT) neurons. In normal mice, 5-HT-immunoreactive fibers are distributed to all cerebellar folia. Following Purkinje cell loss in pcd mice, cerebellar 5-HT-immunoreactive fibers persist. Cerebellar cell suspensions were prepared from embryonic day (E) 11-13 normal mouse embryos and were intraparenchymally grafted into the cerebellum of pcd mutants either directly or after pre-treatment with 5,7-dihydroxytryptamine (5,7-DHT) to selectively remove 5-HT cells of donor origin. The state of Purkinje cells and 5-HT axons was monitored in alternate sections by 28-kDa Ca(2+)-binding protein (CaBP) and 5-HT immunocytochemistry, respectively. Serotonin-immunoreactive axons were seen in the grafts from 5 to 32 days after transplantation. In some of the grafts which had not been pre-treated with 5,7-DHT, a small number of 5-HT-immunoreactive cell bodies was found, indicating that part of the 5-HT fiber innervation of the graft could actually derive from donor cells.(ABSTRACT TRUNCATED AT 250 WORDS)