DARPP-32 Positive Neurons Research Articles

FOXOs modulate proteasome activity in human-induced pluripotent stem cells of Huntington's disease and their derived neural cells.

Although it has been speculated that proteasome dysfunction may contribute to the pathogenesis of Huntington's disease (HD), a devastating neurodegenerative disorder, how proteasome activity is regulated in HD affected stem cells and somatic cells remains largely unclear. To better understand the pathogenesis of HD, we analyzed proteasome activity and the expression of FOXO transcription factors in three wild-type (WT) and three HD induced-pluripotent stem cell (iPSC) lines. HD iPSCs exhibited elevated proteasome activity and higher levels of FOXO1 and FOXO4 proteins. Knockdown of FOXO4 but not FOXO1 expression decreased proteasome activity. Following neural differentiation, the HD-iPSC-derived neural progenitor cells (NPCs) demonstrated lower levels of proteasome activity and FOXO expressions than their WT counterparts. More importantly, overexpression of FOXO4 but not FOXO1 in HD NPCs dramatically enhanced proteasome activity. When HD NPCs were further differentiated into DARPP32-positive neurons, these HD neurons were more susceptible to death than WT neurons and formed Htt aggregates under the condition of oxidative stress. Similar to HD NPCs, HD-iPSC-derived neurons showed reduced proteasome activity and diminished FOXO4 expression compared to WT-iPSC-derived neurons. Furthermore, HD iPSCs had lower AKT activities than WT iPSCs, whereas the neurons derived from HD iPSC had higher AKT activities than their WT counterparts. Inhibiting AKT activity increased both FOXO4 level and proteasome activity, indicating a potential role of AKT in regulating FOXO levels. These data suggest that FOXOs modulate proteasome activity, and thus represents a potentially valuable therapeutic target for HD.

Safety of Striatal Infusion of siRNA in a Transgenic Huntington's Disease Mouse Model.

The immune system In Huntington's disease (HD) is activated and may overreact to some therapies. RNA interference using siRNA lowers mutant huntingtin (mHTT) protein but could increase immune responses. To examine the innate immune response following siRNA infusion into the striatum of wild-type (WT) and HD transgenic (YAC128) mice. siRNAs (2'-O-methyl phosphorothioated) were infused unilaterally into striatum of four month-old WT and YAC128 mice for 28 days. Microglia number and morphology (resting (normal), activated, dystrophic), cytokine levels, and DARPP32-positive neurons were measured in striatum immediately or 14 days post-infusion. Controls included contralateral untreated striatum, and PBS and sham treated striata. The striata of untreated YAC128 mice had significantly fewer resting microglia and more dystrophic microglia than WT mice, but no difference from WT in the proportion of activated microglia or total number of microglia. siRNA infusion increased the total number of microglia in YAC128 mice compared to PBS treated and untreated striata and increased the proportion of activated microglia in WT and YAC128 mice compared to untreated striata and sham treated groups. Cytokine levels were low and siRNA infusion resulted in only modest changes in those levels. siRNA infusion did not change the number of DARPP32-positive neurons. Findings suggest that siRNA infusion may be a safe method for lowering mHTT levels in the striatum in young animals, since treatment does not produce a robust cytokine response or cause neurotoxicity. The potential long-term effects of a sustained increase in total and activated microglia after siRNA infusion in HD mice need to be explored.

Erythropoietin is neuroprotective in a transgenic mouse model of multiple system atrophy

Multiple system atrophy is a rapidly progressive neurodegenerative disorder with a markedly reduced life expectancy. Failure of symptomatic treatment raises an urgent need for disease-modifying strategies. We have investigated the neuroprotective potential of erythropoietin in (proteolipid protein)-α-synuclein transgenic mice exposed to 3-nitropropionic acid featuring multiple system atrophy-like pathology including oligodendroglial α-synuclein inclusions and selective neuronal degeneration. Mice were treated with erythropoietin starting before (early erythropoietin) and after (late erythropoietin) intoxication with 3-nitropropionic acid. Nonintoxicated animals receiving erythropoietin and intoxicated animals treated with saline served as control groups. Behavioral tests included pole test, open field activity, and motor behavior scale. Immunohistochemistry for tyrosine hydroxylase and dopamine and cyclic adenosine monophosphate-regulated phosphoprotein (DARPP-32) was analyzed stereologically. Animals receiving erythropoietin before and after 3-nitropropionic acid intoxication scored significantly lower on the motor behavior scale and they performed better in the pole test than controls with no significant difference between early and late erythropoietin administration. Similarly, rearing scores were worse in 3-nitropropionic acid-treated animals with no difference between the erythropoietin subgroups. Immunohistochemistry revealed significant attenuation of 3-nitropropionic acid-induced loss of tyrosine hydroxylase and DARPP-32 positive neurons in substantia nigra pars compacta and striatum, respectively, in both erythropoietin-treated groups without significant group difference in the substantia nigra. However, at striatal level, a significant difference between early and late erythropoietin administration was observed. In the combined (proteolipid protein)-α-synuclein 3-nitropropionic acid multiple system atrophy mouse model, erythropoietin appears to rescue dopaminergic and striatal gabaergic projection neurons. This effect is associated with improved motor function. Further studies are warranted to develop erythropoietin as a potential interventional therapy in multiple system atrophy.

Colocalization of dopamine receptor subtypes with dopamine and cAMP-regulated phosphoprotein (DARPP-32) in rat brain

In the present study using indirect immunofluorescence immunohistochemistry, co-immunoprecipitation and western blot analysis we determined the colocalization of dopamine receptors 1–5 and dopamine and cAMP-regulated phosphoprotein (DARPP-32) in rat brain cortex and striatum. All five DR subtypes and DARPP-32 were expressed in rat brain cortex and striatum. DARPP-32 positive neurons displayed comparative colocalization with DR1-5. In cingulate cortex, the colocalization of DR subtypes was greatly different from frontal or temporal cortex. D1R is one of the most predominant subtypes which colocalized with DARPP-32 in cortex as well as striatum and followed by D2R, D3R, D4R and D5R. Amongst all DR subtypes D5R was coexpressed the least with DARPP-32 positive neurons. Consistent with immunohistochemical data, western blot analysis also reveals comparable distribution of DR subtypes and DARPP-32 in cortex and striatum. Colocalization studies were also supported by using co-immunoprecipitate assay displaying DARPP-32 expression in DR immunoprecipitate from tissue lysate prepared from cortex and striatum. Taken together our data support receptor specific association of DARPP-32 with DR subtypes that might shed new information in drugs of abuse and pathophysiology of neurodegenerative diseases as well as neuropsychiatric disorders such as schizophrenia.

Neural stem cells for cell replacement therapy for Huntington's disease

The research reported in this thesis focused on the potential of neural precursor cells to provide a suitable source of neurones which can be used in cell replacement strategies for Huntington's disease. Specifically, the parameters affecting the differentiation of these cells into neuronal phenotypes were addressed and increasing the survival of proliferating and differentiating neurones was attempted. In vivo characteristics and the fibre projections of primary and 10 day expanded ENPs was also assessed. The limitations of xenografts in this thesis led to the search for an alternative model system for such experiments. Chapter Three involved an extensive study investigating the effects of a range of concentrations of FGF-2 and EGF on the proliferation and more importantly the neuronal differentiation of murine ENPs over 6 passages in culture, and it was found that the concentration had an effect on the neuronal proportion as well as the neuronal yield of these cultures. Chapter 4 examined the turnover of neuronal precursors in the ENP population cultured in the presence of FGF2 and EGF, using BrdU. The ongoing proliferation of neuronal precursors within ENP cultures was observed and the addition of the growth factors: CNTF, BDNF, HGF and NGF, to enhance the survival of these neurons on differentiation had no effect. Chapter 5 examined the potential of 10 day expanded human striatal ENPs to maintain a striatal like phenotype both in vitro and in vivo in comparison to primary foetal tissue. In vitro after 10 days expansion ENPs differentiated into DARPP-32 positive neurons and this characteristic was maintained in vivo, in a lesion model of HD, albeit to a much lesser extent. This study was limited by the need for ongoing immunosuppression which reduced the life span of the host animal. Chapter 6 investigates further the potential of ENPs. The ability for these cells to send long projections in the host brain and therefore repairing the circuitry lost or damaged as a result of the disease. A four way analysis was carried out examining both alio- and xenograft environments with both primary and 10 day expanded ENPs. Mouse grafts were used to address the allograft environment given that such an experiment is not possible with human tissue and both human and mouse tissue addressed the xenograft environment. To overcome the issues associated with labelling the grafted tissue in the host brain, several techniques were employed, including the use of the GFP transgenic mouse, lentiviral labelling of the cells with the LacZ gene and iontophoretic labelling of the graft with anterograde tracers. ENP grafts were shown to send out longer projections than that of primary tissue although this may be due to migration of the grafted cells. Chapter 7 addresses the issue of immunosuppression of xenografted animals. An alternative model system was explored with the hypothesis being that it would be possible to tolerise the animal in the neonatal period to the xenograft tissue that would subsequently be used for intrastriatal grafting in the adult. Indeed, tolerising the animal resulted in healthy surviving grafts in the adult without the need for daily immunosuppression. Work presented in this thesis contributes some understanding to the biology of neural stem cells and neural xenografts that may ultimately be used for neural transplantation therapies in HD.

Evidence for dopaminergic re-innervation by embryonic allografts in an optimized rat model of the Parkinsonian variant of multiple system atrophy

Embryonic transplantation has been considered as an alternative treatment strategy for drug resistant parkinsonian symptoms in multiple system atrophy. So far our group has created a number of animal models of striatonigral degeneration, the core pathology underlying progressive Parkinsonism associated with multiple system atrophy, as testbed for neurorestaurative and neuroprotective approaches. Using embryonic allografts of either nigral, striatal, or combined nigro-striatal tissue we were able to consistently show graft survival in a denervated and lesioned striatum as well as improvement of rotational behaviour. However, due to severe lesions of the striatum and the chosen time window of 3–6 weeks between lesion and grafting, severe gliosis led to demarcation of the graft and prevented re-innervation of the remaining adult striatum. The aim of the present study was to modify our “double toxin-double lesion” rat model by reducing the dose of quinolinic acid injected into the striatum from 150 to 75 nmol and shortening the interval between lesion and grafting to 1–2 weeks. Injection of 75 nmol quinolinic acid still led to a significant reduction of DARPP-32 positive neurons and volume in the striatum. Analysis of embryonic mesencephalic grafts revealed survival of dopaminergic neurons and outgrowth of fibres re-innervating the adult striatum. Rotation behaviour was improved in the graft group. Considering embryonic transplantation a possible future antiparkinson therapeutic intervention in multiple system atrophy patients our data stress the necessity of optimal patient selection, i.e. early stage disease with limited striatal dysfunction.

The development of intracerebral cell-suspension implants is influenced by the grafting medium

The effect of preparing and grafting embryonic striatal and nigral tissue in four different media was evaluated in vitro and in vivo. The proportion of TH-positive and DARPP-32–positive neurons was determined after 2 days in vitro in standard culture medium following preparation in the different media. The effects were more marked for striatal neurons where DARPP-32 expression in tissue prepared in HBSS was poor compared to other media. TH expression was unaffected by the preparation medium. Striatal grafts derived from tissue prepared and grafted in HBSS were smaller, with fewer DARPP-32 cells, compared to other media. Survival of grafts in combined HBSS and DMEM was very poor. Graft volume and TH cell content was enhanced in tissue prepared in DMEM. These results suggest that preparation protocols optimized for one type of embryonic neuronal population do not necessarily transfer to other neuronal populations.

The development of intracerebral cell-suspension implants is influenced by the grafting medium.

The effect of preparing and grafting embryonic striatal and nigral tissue in four different media was evaluated in vitro and in vivo. The proportion of TH-positive and DARPP-32-positive neurons was determined after 2 days in vitro in standard culture medium following preparation in the different media. The effects were more marked for striatal neurons where DARPP-32 expression in tissue prepared in HBSS was poor compared to other media. TH expression was unaffected by the preparation medium. Striatal grafts derived from tissue prepared and grafted in HBSS were smaller, with fewer DARPP-32 cells, compared to other media. Survival of grafts in combined HBSS and DMEM was very poor. Graft volume and TH cell content was enhanced in tissue prepared in DMEM. These results suggest that preparation protocols optimized for one type of embryonic neuronal population do not necessarily transfer to other neuronal populations.

Development of intrastriatal striatal grafts and their afferent innervation from the host

The morphological maturation of cell suspension grafts of fetal striatal tissue (obtained from 14–15-day-old rat fetuses) was followed from two days to eight weeks after implantation into intact and ibotenic acid-lesioned striata of adult rats. The development of host afferent innervation of the grafts from the substantia nigra (tyrosine hydroxylase immunoreactive), mesencephalic raphe (serotonin immuno-reactive), and the frontal cortex (anterogradely labelled with Phaseolus vulgaris leucoagglutinin) were revealed by immunohistochemistry. During the first weeks post-grafting, the striatal implants consisted of a mixture of mature- and immature-looking cell clusters. Grafts implanted into ibotenic acid-lesioned striatum grew rapidly (about five-fold) in volume over the first week. The areas of immature (probably proliferating) cells gradually disappeared, and by six to eight weeks the grafts had a fully mature appearance with patches of neurons which stained densely for DARPP-32 (i.e. were striatum-like) embedded within areas of essentially DARPP-32-negative (i.e. non-striatum-like) tissue. Peripheral clusters of grafted cells gradually intermingled with nearby areas of the surrounding lesioned host, and already by two to four days after implantation, coarse and densely immunoreactive host fibres from the substantia nigra, mesencephalic raphe and frontal cortex were present within the grafts. By four to five days the first DARPP-32-immunoreactive neurons appeared in patches within the mature portions of the grafts, and one to two days later the tyrosine hydroxylase-positive fibres began to sprout thin axons selectively within the DARPP-32-positive patches. Similarly, the serotonergic and cortical fibres in the grafts increased in number over the next two weeks, but they showed no preference for the DARPP-32-positive regions. Rich terminal networks were established by two to three weeks post-grafting, and by six to eight weeks the nigral, raphe and cortical afferents had reached terminal densities similar to those seen previously in long-term surviving grafts. Grafts implanted into dopamine-denervated hosts showed a normal morphological maturation of both DARPP-32-positive and -negative areas, although no tyrosine hydroxylase-positive innervation appeared within the grafts. Grafts implanted into non-lesioned striata did not grow beyond their initial size. The implanted cells showed less intermingling with the surrounding host striatum, thus resulting in sharply delineated graft-host borders. DARPP-32-positive patches developed, but they were smaller in size and generally present only in the most peripheral graft portions. Host dopaminergic, serotonergic and cortical afferents occurred from the first week, although more restricted to peripheral portions, and with lower densities than in grafts implanted into lesioned hosts. Control grafts of fetal cerebellar tissue, implanted into lesioned striatum, intermingled with coarse fibres from the various host afferent systems within the first week. However, there appeared to be less sprouting of thin axons from the various host afferents. Only scattered and weakly labelled DARPP-32 positive neurons occurred, and the tyrosine hydroxylase-positive fibres did not concentrate into dense patches. The results indicate that intrastriatal fetal striatal grafts develop with a time-course which is only slightly delayed compared to their normal development, and that functional host-to-graft integration may occur as early as two to three weeks after implantation. This integration occurred in two, partly overlapping, phases: first a rapid growth and non-specific intermingling of the implanted cells with the lesioned host and its target-deprived afferents within the first eight to ten days, followed by a second phase of host axonal sprouting and formation of terminal-like fibre networks within the implants, which was complete within six to eight weeks.

DARPP‐32, a phosphoprotein enriched in dopaminoceptive neurons bearing dopamine D1 receptors: DIstribution in the cerebral cortex of the newborn and adult rhesus monkey

DARPP-32, a dopamine (DA) and cAMP-regulated phosphoprotein, is associated with dopaminoceptive neurons bearing D-1 receptors in the basal ganglia. The present study addressed the distribution of DARPP-32 in the primate cerebral cortex and its putative association with D-1 receptor laden cells in this structure. DARPP-32-like immunoreactive (LIR) neurons were examined in the cerebral cortex of 3-day-old (P3), 6-week-old (P42), and adult rhesus monkeys. In the younger cases, a large number of DARPP-32 positive neurons, with the morphological characteristics of pyramidal cells, were observed throughout the cortex, in layers V-VI, and to a lesser extent in layer II and uppermost layer III. In the parietal, insular, temporal, and occipital cortices, DARPP-32 positive neurons were arranged in a monolayer in layer Va. They were often clustered in small groups with a bundling of their dendrites. In the primary motor cortex, Betz cells were among the labeled population. In the association and somatosensory areas, the basal dendrites of DARPP-32 positive neurons and the prominent tufting of their apical dendrites in layer I contributed to an essential bilaminar pattern resembling the distribution reported for DA afferents and D-1 receptors in these areas. The prominence and widespread distribution of DARPP-32 positive neurons in layer V may be a specialization of primate cortex since such cells are found only in restricted locations in rodents. The literature on the connections of the cerebral cortex suggests that a large number of the DARPP-32 positive neurons in layer VI and perhaps even in layer Va may be corticothalamic neurons. An important developmental observation was the presence of DARPP-32-LIR neurons in the white matter. They were prominent in the neonates but could not be seen in the adult. Their location as well as their type and shape were reminiscent of interstitial neurons. In the adult monkeys, the distribution of DARPP-32-LIR neurons was more circumscribed: they were numerous in the ventral temporal gyrus and in areas related to the limbic system: caudal orbitofrontal cortex, insula, temporal pole, entorhinal, and anterior cingulate cortex. Weak labeling was detected in layer Va of the superior temporal and parietal cortex, in some prefrontal areas (10, 13, and medial 9), and in the premotor and supplementary motor cortex; in adults, unlike neonates, few DARPP-32-LIR neurons were present in the dorsolateral prefrontal cortex, the primary motor or the primary visual or prestriate cortices.(ABSTRACT TRUNCATED AT 400 WORDS)