Purkinje Cell Degeneration Mice Research Articles

Intracerebellar injection of monocytic immature myeloid cells prevents the adverse effects caused by stereotactic surgery in a model of cerebellar neurodegeneration

BackgroundMyeloid-derived suppressor cells (MDSCs) constitute a recently discovered bone-marrow-derived cell type useful for dealing with neuroinflammatory disorders. However, these cells are only formed during inflammatory conditions from immature myeloid cells (IMCs) that acquire immunosuppressive activity, thus being commonly gathered from diseased animals. Then, to obtain a more clinically feasible source, we characterized IMCs directly derived from healthy bone marrow and proved their potential immunosuppressive activity under pathological conditions in vitro. We then explored their neuroprotective potential in a model of human cerebellar ataxia, the Purkinje Cell Degeneration (PCD) mouse, as it displays a well-defined neurodegenerative and neuroinflammatory process that can be also aggravated by invasive surgeries.MethodsIMCs were obtained from healthy bone marrow and co-cultured with activated T cells. The proliferation and apoptotic rate of the later were analyzed with Tag-it Violet. For in vivo studies, IMCs were transplanted by stereotactic surgery into the cerebellum of PCD mice. We also used sham-operated animals as controls of the surgical effects, as well as their untreated counterparts. Motor behavior of mice was assessed by rotarod test. The Purkinje cell density was measured by immunohistochemistry and cell death assessed with the TUNEL technique. We also analyzed the microglial phenotype by immunofluorescence and the expression pattern of inflammation-related genes by qPCR. Parametric tests were applied depending on the specific experiment: one or two way ANOVA and Student’s T test.ResultsIMCs were proven to effectively acquire immunosuppressive activity under pathological conditions in vitro, thus acting as MDSCs. Concerning in vivo studios, sham-operated PCD mice suffered detrimental effects in motor coordination, Purkinje cell survival and microglial activation. After intracranial administration of IMCs into the cerebellum of PCD mice, no special benefits were detected in the transplanted animals when compared to untreated mice. Nonetheless, this transplant almost completely prevented the impairments caused by the surgery in PCD mice, probably by the modulation of the inflammatory patterns.ConclusionsOur work comprise two main translational findings: (1) IMCs can be directly used as they behave as MDSCs under pathological conditions, thus avoiding their gathering from diseased subjects; (2) IMCs are promising adjuvants when performing neurosurgery.

Reduction of Microvessel Number and Length in the Cerebellum of Purkinje Cell Degeneration Mice.

Degenerative effects of nerve tissues are often accompanied by changes in vascularization. In this regard, knowledge about hereditary cerebellar degeneration is limited. In this study, we compared the vascularity of the individual cerebellar components of 3-month-old wild-type mice (n = 8) and Purkinje cell degeneration (pcd) mutant mice, which represent a model of hereditary cerebellar degeneration (n = 8). Systematic random samples of tissue sections were processed, and laminin was immunostained to visualize microvessels. A computer-assisted stereology system was used to quantify microvessel parameters including total number, total length, and associated densities in cerebellar layers. Our results in pcd mice revealed a 45% (p < 0.01) reduction in the total volume of the cerebellum, a 28% (p < 0.05) reduction in the total number of vessels and a lower total length, approaching 50% (p < 0.001), compared to the control mice. In pcd mutants, cerebellar degeneration is accompanied by significant reduction in the microvascular network that is proportional to the cerebellar volume reduction therefore does not change density of in the cerebellar gray matter of pcd mice.

The Selective Loss of Purkinje Cells Induces Specific Peripheral Immune Alterations.

The progression of neurodegenerative diseases is reciprocally associated with impairments in peripheral immune responses. We investigated different contexts of selective neurodegeneration to identify specific alterations of peripheral immune cells and, at the same time, discover potential biomarkers associated to this pathological condition. Consequently, a model of human cerebellar degeneration and ataxia -the Purkinje Cell Degeneration (PCD) mouse- has been employed, as it allows the study of different processes of selective neuronal death in the same animal, i.e., Purkinje cells in the cerebellum and mitral cells in the olfactory bulb. Infiltrated leukocytes were studied in both brain areas and compared with those from other standardized neuroinflammatory models obtained by administering either gamma radiation or lipopolysaccharide. Moreover, both myeloid and lymphoid splenic populations were analyzed by flow cytometry, focusing on markers of functional maturity and antigen presentation. The severity and type of neural damage and inflammation affected immune cell infiltration. Leukocytes were more numerous in the cerebellum of PCD mice, being located predominantly within those cerebellar layers mostly affected by neurodegeneration, in a completely different manner than the typical models of induced neuroinflammation. Furthermore, the milder degeneration of the olfactory bulb did not foster leukocyte attraction. Concerning the splenic analysis, in PCD mice we found: (1) a decreased percentage of several myeloid cell subsets, and (2) a reduced mean fluorescence intensity in those myeloid markers related to both antigen presentation and functional maturity. In conclusion, the selective degeneration of Purkinje cells triggers a specific effect on peripheral immune cells, fostering both attraction and functional changes. This fact endorses the employment of peripheral immune cell populations as concrete biomarkers for monitoring different neuronal death processes.

The Childhood-Onset Neurodegeneration with Cerebellar Atrophy (CONDCA) Disease Caused by AGTPBP1 Gene Mutations: The Purkinje Cell Degeneration Mouse as an Animal Model for the Study of this Human Disease.

Recent reports have identified rare, biallelic damaging variants of the AGTPBP1 gene that cause a novel and documented human disease known as childhood-onset neurodegeneration with cerebellar atrophy (CONDCA), linking loss of function of the AGTPBP1 protein to human neurodegenerative diseases. CONDCA patients exhibit progressive cognitive decline, ataxia, hypotonia or muscle weakness among other clinical features that may be fatal. Loss of AGTPBP1 in humans recapitulates the neurodegenerative course reported in a well-characterised murine animal model harbouring loss-of-function mutations in the AGTPBP1 gene. In particular, in the Purkinje cell degeneration (pcd) mouse model, mutations in AGTPBP1 lead to early cerebellar ataxia, which correlates with the massive loss of cerebellar Purkinje cells. In addition, neurodegeneration in the olfactory bulb, retina, thalamus and spinal cord were also reported. In addition to neurodegeneration, pcd mice show behavioural deficits such as cognitive decline. Here, we provide an overview of what is currently known about the structure and functional role of AGTPBP1 and discuss the various alterations in AGTPBP1 that cause neurodegeneration in the pcd mutant mouse and humans with CONDCA. The sequence of neuropathological events that occur in pcd mice and the mechanisms governing these neurodegenerative processes are also reported. Finally, we describe the therapeutic strategies that were applied in pcd mice and focus on the potential usefulness of pcd mice as a promising model for the development of new therapeutic strategies for clinical trials in humans, which may offer potential beneficial options for patients with AGTPBP1 mutation-related CONDCA.

In vivo 5-ethynyluridine (EU) labelling detects reduced transcription in Purkinje cell degeneration mouse mutants, but can itself induce neurodegeneration

Fluorescent staining of newly transcribed RNA via metabolic labelling with 5-ethynyluridine (EU) and click chemistry enables visualisation of changes in transcription, such as in conditions of cellular stress. Here, we tested whether EU labelling can be used to examine transcription in vivo in mouse models of nervous system disorders. We show that injection of EU directly into the cerebellum results in reproducible labelling of newly transcribed RNA in cerebellar neurons and glia, with cell type-specific differences in relative labelling intensities, such as Purkinje cells exhibiting the highest levels. We also observed EU-labelling accumulating into cytoplasmic inclusions, indicating that EU, like other modified uridines, may introduce non-physiological properties in labelled RNAs. Additionally, we found that EU induces Purkinje cell degeneration nine days after EU injection, suggesting that EU incorporation not only results in abnormal RNA transcripts, but also eventually becomes neurotoxic in highly transcriptionally-active neurons. However, short post-injection intervals of EU labelling in both a Purkinje cell-specific DNA repair-deficient mouse model and a mouse model of spinocerebellar ataxia 1 revealed reduced transcription in Purkinje cells compared to controls. We combined EU labelling with immunohistology to correlate altered EU staining with pathological markers, such as genotoxic signalling factors. These data indicate that the EU-labelling method provided here can be used to identify changes in transcription in vivo in nervous system disease models.

Oleoylethanolamide Delays the Dysfunction and Death of Purkinje Cells and Ameliorates Behavioral Defects in a Mouse Model of Cerebellar Neurodegeneration.

Oleoylethanolamide (OEA) is an endocannabinoid that has been proposed to prevent neuronal damage and neuroinflammation. In this study, we evaluated the effects of OEA on the disruption of both cerebellar structure and physiology and on the behavior of Purkinje cell degeneration (PCD) mutant mice. These mice exhibit cerebellar degeneration, displaying microtubule alterations that trigger the selective loss of Purkinje cells and consequent behavioral impairments. The effects of different doses (1, 5, and 10mg/kg, i.p.) and administration schedules (chronic and acute) of OEA were assessed at the behavioral, histological, cellular, and molecular levels to determine the most effective OEA treatment regimen. Our in vivo results demonstrated that OEA treatment prior to the onset of the preneurodegenerative phase prevented morphological alterations in Purkinje neurons (the somata and dendritic arbors) and decreased Purkinje cell death. This effect followed an inverted U-shaped time-response curve, with acute administration on postnatal day 12 (10mg/kg, i.p.) being the most effective treatment regimen tested. Indeed, PCD mice that received this specific OEA treatment regimen showed improvements in motor, cognitive and social functions, which were impaired in these mice. Moreover, these in vivo neuroprotective effects of OEA were mediated by the PPARα receptor, as pretreatment with the PPARα antagonist GW6471 (2.5mg/kg, i.p.) abolished them. Finally, our in vitro results suggested that the molecular effect of OEA was related to microtubule stability and structure since OEA administration normalized some alterations in microtubule features in PCD-like cells. These findings provide strong evidence supporting the use of OEA as a pharmacological agent to limit severe cerebellar neurodegenerative processes.

Secretagogin expression in the mouse olfactory bulb under sensory impairments

The interneurons of the olfactory bulb (OB) are characterized by the expression of different calcium-binding proteins, whose specific functions are not fully understood. This is the case of one of the most recently discovered, the secretagogin (SCGN), which is expressed in interneurons of the glomerular and the granule cell layers, but whose function in the olfactory pathway is still unknown. To address this question, we examined the distribution, generation and activity of SCGN-positive interneurons in the OB of two complementary models of olfactory impairments: Purkinje Cell Degeneration (PCD) and olfactory-deprived mice. Our results showed a significant increase in the density of SCGN-positive cells in the inframitral layers of olfactory-deprived mice as compared to control animals. Moreover, BrdU analyses revealed that these additional SCGN-positive cells are not newly formed. Finally, the neuronal activity, estimated by c-Fos expression, increased in preexisting SCGN-positive interneurons of both deprived and PCD mice -being higher in the later- in comparison with control animals. Altogether, our results suggest that the OB possesses different compensatory mechanisms depending on the type of alteration. Particularly, the SCGN expression is dependent of olfactory stimuli and its function may be related to a compensation against a reduction in sensory inputs.

Daily bone marrow cell transplantations for the management of fast neurodegenerative processes.

Cell therapy has been proven to be a promising treatment for fighting neurodegenerative diseases. As neuronal replacement presents undeniable complications, the neuroprotection of live neurons arises as the most suitable therapeutic approach. Accordingly, the earlier the diagnosis and treatment, the better the prognosis. However, these diseases are commonly diagnosed when symptoms have already progressed towards an irreversible degenerative stage. This problem is especially dramatic when neurodegeneration is aggressive and rapidly progresses. One of the most interesting approaches for neuroprotection is the fusion between healthy bone marrow-derived cells and neurons, as the former can provide the latter with regular/protective genes without harming brain parenchyma. So far, this phenomenon has only been identified in Purkinje cells, whose death is the cause of different diseases like cerebellar ataxias. Here we have employed a model of aggressive cerebellar neurodegeneration, the Purkinje Cell Degeneration mouse, to optimize a cell therapy based on bone marrow-derived cell and cell fusion. Our findings show that the substitution of bone marrow in diseased animals by healthy bone marrow, even prior to the onset of neurodegeneration, is not fast enough to stop neuronal loss in time. Conversely, avoiding bone marrow replacement and ensuring a regular supply of healthy cells through continuous, daily transplants, the neurodegenerative milieu of PCD is enough to attract those transplanted elements. Furthermore, in the most affected cerebellar regions, more than a half of surviving neurons undergo a process of cell fusion. Therefore, this method deserves consideration as a means to impede neuronal cell death.

Bone marrow transplantation improves motor activity in a mouse model of ataxia.

Ataxias are locomotor disorders that can have an origin both neural and muscular, although both impairments are related. Unfortunately, ataxia has no cure, and the current therapies are aimed at motor re-education or muscular reinforcement. Nevertheless, cell therapy is becoming a promising approach to deal with incurable neural diseases, including neuromuscular ataxias. Here, we have used a model of ataxia, the Purkinje Cell Degeneration (PCD) mutant mouse, to study the effect of healthy (wild-type) bone marrow transplantation on the restoration of defective mobility. Bone marrow transplants (from both mutant and healthy donors) were performed in wild-type and PCD mice. Then, a wide battery of behavioural tests was employed to determine possible motor amelioration in mutants. Finally, cerebellum, spinal cord, and muscle were analysed to study the integration of the transplant-derived cells and the origin of the behavioural changes. Our results demonstrated that the transplant of wild-type bone marrow restores the mobility of PCD mice, increasing their capabilities of movement (52-100% of recovery), exploration (20-71% of recovery), speed (35% of recovery), and motor coordination (25% of recovery). Surprisingly, our results showed that bone marrow transplant notably improves the skeletal muscle structure, which is severely damaged in the mutants, rather than ameliorating the central nervous system. Although a multimodal effect of the transplant is not discarded, muscular improvements appear to be the basis of this motor recovery. Furthermore, the results from our study indicate that bone marrow stem cell therapy can be a safe and effective alternative for dealing with movement disorders such as ataxias.

Linking Essential Tremor to the Cerebellum-Animal Model Evidence.

In this review, we hope to stimulate interest in animal models as opportunities to understand tremor mechanisms within the cerebellar system. We begin by considering the harmaline model of essential tremor (ET), which has ET-like anatomy and pharmacology. Harmaline induces the inferior olive (IO) to burst fire rhythmically, recruiting rhythmic activity in Purkinje cells (PCs) and deep cerebellar nuclei (DCN). This model has fostered the IO hypothesis of ET, which postulates that factors that promote excess IO, and hence PC complex spike synchrony, also promote tremor. In contrast, the PC hypothesis postulates that partial PC cell loss underlies tremor of ET. We describe models in which chronic partial PC loss is associated with tremor, such as the Weaver mouse, and others with PC loss that do not show tremor, such as the Purkinje cell degeneration mouse. We postulate that partial PC loss with tremor is associated with terminal axonal sprouting. We then discuss tremor that occurs with large lesions of the cerebellum in primates. This tremor has variable frequency and is an ataxic tremor not related to ET. Another tremor type that is not likely related to ET is tremor in mice with mutations that cause prolonged synaptic GABA action. This tremor is probably due to mistiming within cerebellar circuitry. In the final section, we catalog tremor models involving neurotransmitter and ion channel perturbations. Some appear to be related to the IO hypothesis of ET, while in others tremor may be ataxic or due to mistiming. In summary, we offer a tentative framework for classifying animal action tremor, such that various models may be considered potentially relevant to ET, subscribing to IO or PC hypotheses, or not likely relevant, as with mistiming or ataxic tremor. Considerable further research is needed to elucidate the mechanisms of tremor in animal models.

Transplantation and Stem Cell Therapy for Cerebellar Degenerations.

Stem cell-based and regenerative therapy may become a hopeful treatment for neurodegenerative diseases including hereditary cerebellar degenerations. Neurotransplantation therapy mainly aims to substitute lost cells, but potential effects might include various mechanisms including nonspecific trophic effects and stimulation of endogenous regenerative processes and neural plasticity. Nevertheless, currently, there remain serious limitations. There is a wide spectrum of human hereditary cerebellar degenerations as well as numerous cerebellar mutant mouse strains that serve as models for the development of effective therapy. By now, transplantation has been shown to ameliorate cerebellar function, e.g. in Purkinje cell degeneration mice, Lurcher mutant mice and mouse models of spinocerebellar ataxia type 1 and type 2 and Niemann-Pick disease type C. Despite the lack of direct comparative studies, it appears that there might be differences in graft development and functioning between various types of cerebellar degeneration. Investigation of the relation of graft development to specific morphological, microvascular or biochemical features of the diseased host tissue in various cerebellar degenerations may help to identify factors determining the fate of grafted cells and potential of their functional integration.

Ischemia-induced autophagy contributes to neurodegeneration in cerebellar Purkinje cells in the developing rat brain and in primary cortical neurons in vitro

Increased autophagy/mitophagy is thought to contribute to cerebellar dysfunction in Purkinje cell degeneration mice. Intriguingly, cerebellar Purkinje cells are highly vulnerable to hypoxia–ischemia (HI), related at least in part to their high metabolic activity. Whether or not excessive or supraphysiologic autophagy plays a role in Purkinje cell susceptibility to HI is unknown. Accordingly, we evaluated the role of autophagy in the cerebellum after global ischemia produced by asphyxial cardiac arrest in postnatal day (PND) 16–18 rats, using siRNA-targeted inhibition of Atg7, necessary for microtubule-associated protein light chain 3-II (LC3-II) and Atg12–Atg5 complex formation. Two days before a 9min asphyxial cardiac arrest or sham surgery, Atg7 or control siRNA was injected intracisternally to target the cerebellum. Treatment with Atg7 siRNA: 1) reduced Atg7 protein expression in the cerebellum by 56%; 2) prevented the typical ischemia-induced formation of LC3-II in the cerebellum 24h after asphyxial cardiac arrest; 3) improved performance on the beam-balance apparatus on days 1–5; and 4) increased calbindin-labeled Purkinje cell survival assessed on day 14. Improved Purkinje cell survival was more consistent in female vs. male rats, and improved beam-balance performance was only seen in female rats. Similar responses to Atg7 siRNA i.e. reduced autophagy and neurodegeneration vs. control siRNA were seen when exposing sex-segregated green fluorescent protein-LC3 tagged mouse primary cortical neurons to oxygen glucose deprivation in vitro. Thus, inhibition of autophagy after global ischemia in PND 16–18 rats leads to increased survival of Purkinje cells and improved motor performance in a sex-dependent manner.

Hereditary cerebellar degeneration and stem-cell based therapy

Hereditary cerebellar ataxias are a wide group of progressive, degenerative diseases, which manifest as lack of control and coordination of voluntary movements due to progressive loss of Purkinje cells and other cell types in the cerebellum. Cerebellar degeneration could affect people of any age, is often diagnosed after symptoms start to manifest and no prevention and no causal and really effective therapy is available today. There is high variability in pathogenesis in human cerebellar degenerations and high variability in onset and progress of the disease. Though the basal manifestation of all cerebellar degenerations is similar as it is related to dysfunction of the cerebellum, there are some differences between individual types of them and also some variability in the frame of one disease making the clinical diagnosis difficult. The manifestation of the mutation could be also modified by the genetic background. For all of that, individual approach of treatment is necessary. There is a vide spectrum of mouse models of genetically determined cerebellar degenerations including both spontaneous mutants and transgenic mice, some of which are available in several mouse strains. This situation is analogous to variability of human hereditary cerebellar disorders and genetic heterogeneity of human population. Mouse models can be used for investigation of efficiency of experimental therapy in individual types of cerebellar degenerations and for studying of the effect of genetic background on the pathological process. Such research could allow identifying factors determining progress of the disease and influencing the effect of the therapy. This is necessary to choose the optimal treatment for each individual patient and for development of new therapeutic methods. One of therapies which could benefit from such research is stem cell and neurotransplantation therapy. So far it seems promising in slowing down progress, but it might even mitigate neurological deficit caused by the cerebellar degeneration. Nevertheless, as experiments in mice shoved different types of grafts have different effects. For example mesenchymal stem cells prevent neurodegeneration progression, but do not reverse already developed cell loss and neurological deficit. Transplanted suspension of embryonic cerebellar cells generates new graft-derived Purkinje cells in Purkinje cell degeneration mice and improvement of motor function was described in them. On the other hand, in Lurcher mice functional benefit of cerebellar tissue transplantation has not been achieved yet. In addition, cultivated embryonic neural stem cells survive well in the host cerebellum and despite they do not create any new Purkinje cells they have been shown to rescue Purkinje cells in a mouse model of Niemann-Pick disease. Genetically defined mouse strains with different mutations causing neurodegenerative diseases and different mouse strain backgrounds for the same mutations and analysis of their genome and proteome would be valuable tool to identify genetic markers and factors important for assessment of prognosis and management of appropriate stem-cell therapy and for development of new therapeutic approaches based on the principles of personalized medicine.

A mitochondrial location for haemoglobins—Dynamic distribution in ageing and Parkinson's disease

Haemoglobins are iron-containing proteins that transport oxygen in the blood of most vertebrates. The mitochondrion is the cellular organelle which consumes oxygen in order to synthesise ATP. Mitochondrial dysfunction is implicated in neurodegeneration and ageing. We find that α and β haemoglobin (Hba and Hbb) proteins are altered in their distribution in mitochondrial fractions from degenerating brain. We demonstrate that both Hba and Hbb are co-localised with the mitochondrion in mammalian brain. The precise localisation of the Hbs is within the inner membrane space and associated with inner mitochondrial membrane. Relative mitochondrial to cytoplasmic ratios of Hba and Hbb show changing distributions of these proteins during the process of neurodegeneration in the pcd5j mouse brain. A significant difference in mitochondrial Hba and Hbb content in the mitochondrial fraction is seen at 31days after birth, this corresponds to a stage when dynamic neuronal loss is measured to be greatest in the Purkinje Cell Degeneration mouse. We also report changes in mitochondrial Hba and Hbb levels in ageing brain and muscle. Significant differences in mitochondrial Hba and Hbb can be seen when comparing aged brain to muscle, suggesting tissue specific functions of these proteins in the mitochondrion. In muscle there are significant differences between Hba levels in old and young mitochondria. To understand whether the changes detected in mitochondrial Hbs are of clinical significance, we examined Parkinson's disease brain, immunohistochemistry studies suggest that cell bodies in the substantia nigra accumulate mitochondrial Hb. However, western blotting of mitochondrial fractions from PD and control brains indicates significantly less Hb in PD brain mitochondria. One explanation could be a specific loss of cells containing mitochondria loaded with Hb proteins. Our study opens the door to an examination of the role of Hb function, within the context of the mitochondrion—in health and disease.

Differential glial activation during the degeneration of Purkinje cells and mitral cells in the PCD mutant mice

Purkinje Cell Degeneration (PCD) mice harbor a nna1 gene mutation which leads to an early and rapid degeneration of Purkinje cells (PC) between the third and fourth week of age. This mutation also underlies the death of mitral cells (MC) in the olfactory bulb (OB), but this process is slower and longer than in PC. No clear interpretations supporting the marked differences in these neurodegenerative processes exist. Growing evidence suggests that either beneficial or detrimental effects of gliosis in damaged regions would underlie these divergences. Here, we examined the gliosis occurring during PC and MC death in the PCD mouse. Our results demonstrated different glial reactions in both affected regions. PC disappearance stimulated a severe gliosis characterized by strong morphological changes, enhanced glial proliferation, as well as the release of pro-inflammatory mediators. By contrast, MC degeneration seems to promote a more attenuated glial response in the PCD OB compared with that of the cerebellum. Strikingly, cerebellar oligodendrocytes died by apoptosis in the PCD, whereas bulbar ones were not affected. Interestingly, the level of nna1 mRNA under normal conditions was higher in the cerebellum than in the OB, probably related to a faster neurodegeneration and stronger glial reaction in its absence. The glial responses may thus influence the neurodegenerative course in the cerebellum and OB of the mutant mouse brain, providing harmful and beneficial microenvironments, respectively.

Bone Marrow Cell Transplantation Restores Olfaction in the Degenerated Olfactory Bulb

Bone marrow contains heterogeneous cell types including end-lineage cells, committed tissue progenitors, and multipotent stem/progenitor cells. The immense plasticity of bone marrow cells allows them to populate diverse tissues such as the encephalon, and give rise to a variety of cell types. This unique plasticity makes bone marrow-derived cells good candidates for cell therapy aiming at restoring impaired brain circuits. In the present study, bone marrow cells were transplanted into P20 mice that exhibit selective olfactory degeneration in adulthood between P60 and P150. These animals, the so-called Purkinje Cell Degeneration (PCD) mutant mice, suffer from a progressive and specific loss of a subpopulation of principal neurons of the olfactory bulb, the mitral cells (MCs), sparing the other principal neurons, the tufted cells. As such, PCD mice constitute an interesting model to evaluate the specific role of MCs in olfaction and to test the restorative function of transplanted bone marrow-derived cells. Using precision olfactometry, we revealed that mutant mice lacking MCs exhibited a deficit in odorant detection and discrimination. Remarkably, the transplantation of wild-type bone marrow-derived cells into irradiated PCD mutant mice generated a large population of microglial cells in the olfactory bulb and reduced the degenerative process. The alleviation of MC loss in transplanted mice was accompanied by functional recovery witnessed by significantly improved olfactory detection and enhanced odor discrimination. Together, these data suggest that: (1) bone marrow-derived cells represent an effective neuroprotective tool to restore degenerative brain circuits, and (2) MCs are necessary to encode odor concentration and odor identity in the mouse olfactory bulb.

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.

Bone Marrow Contributes Simultaneously to Different Neural Types in the Central Nervous System through Different Mechanisms of Plasticity

Many studies have reported the contribution of bone marrow-derived cells (BMDC) to the CNS, raising the possibility of using them as a new source to repair damaged brain tissue or restore neuronal function. This process has mainly been investigated in the cerebellum, in which a degenerative microenvironment has been suggested to be responsible for its modulation. The present study further analyzes the contribution of BMDC to different neural types in other adult brain areas, under both physiological and neurodegenerative conditions, together with the mechanisms of plasticity involved. We grafted genetically marked green fluorescent protein/Cre bone marrow in irradiated recipients: a) the PCD (Purkinje Cell Degeneration) mutant mice, suffering a degeneration of specific neuronal populations at different ages, and b) their corresponding healthy controls. These mice carried the conditional lacZ reporter gene to allow the identification of cell fusion events. Our results demonstrate that BMDC mainly generate microglial cells, although to a lesser extent a clear formation of neuronal types also exists. This neuronal recruitment was not increased by the neurodegenerative processes occurring in PCD mice, where BMDC did not contribute to rescuing the degenerated neuronal populations either. However, an increase in the number of bone marrow-derived microglia was found along the life span in both experimental groups. Six weeks after transplantation more bone marrow-derived microglial cells were observed in the olfactory bulb of the PCD mice compared to the control animals, where the degeneration of mitral cells was in process. In contrast, this difference was not observed in the cerebellum, where Purkinje cell degeneration had been completed. These findings demonstrated that the degree of neurodegenerative environment can foster the recruitment of neural elements derived from bone marrow, but also provide the first evidence that BMDC can contribute simultaneously to different encephalic areas through different mechanisms of plasticity: cell fusion for Purkinje cells and differentiation for olfactory bulb interneurons.

Mitochondrial Dysfunction in NnaD Mutant Flies and Purkinje Cell Degeneration Mice Reveals a Role for Nna Proteins in Neuronal Bioenergetics

The Purkinje cell degeneration (pcd) mouse is a recessive model of neurodegeneration, involving cerebellum and retina. Purkinje cell death in pcd is dramatic, as >99% of Purkinje neurons are lost in 3 weeks. Loss of function of Nna1 causes pcd, and Nna1 is a highly conserved zinc carboxypeptidase. To determine the basis of pcd, we implemented a two-pronged approach, combining characterization of loss-of-function phenotypes of the Drosophila Nna1 ortholog (NnaD) with proteomics analysis of pcd mice. Reduced NnaD function yielded larval lethality, with survivors displaying phenotypes that mirror disease in pcd. Quantitative proteomics revealed expression alterations for glycolytic and oxidative phosphorylation enzymes. Nna proteins localize to mitochondria, loss of NnaD/Nna1 produces mitochondrial abnormalities, and pcd mice display altered proteolytic processing of Nna1 interacting proteins. Our studies indicate that Nna1 loss of function results in altered bioenergetics and mitochondrial dysfunction.

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.