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We have reported recently that the microtubule-associated protein tau is phosphorylated in vitro by Akt, an important kinase in anti-apoptotic signaling regulated by insulin and growth factors. We also established that Akt phosphorylates tau separately at T212 and S214, two sites previously shown to be phosphorylated by glycogen synthase kinase 3beta (GSK3beta) and protein kinase A (PKA), respectively. In the present studies, we examined the relationship between Akt and T212/S214 in primary cultures of human neurons and astrocytes, and evaluated the contribution of two other kinases. In intact cells, we found a very low content of active (phospho-S473) form of Akt. We also found a low content of phospho-S214 but not phospho-T212 of tau, suggesting that only phospho-S212 may depend on Akt activity in situ. We upregulated Akt activity using two experimental models: treatment with a protein phosphatase inhibitor, okadaic acid, and transfection with a constitutively active Akt gene construct (c-Akt). Under these conditions, phosphorylation of tau at T212 and S214 was regulated independently, with little change or downregulation of phospho-T212 and dynamic upregulation of phospho-S214. Our studies revealed that Akt may influence the phospho-S214 content in a meaningful manner. They also revealed that PKA may only partially contribute to the phosphorylation of S214. In comparison, okadaic acid treatment severely depleted the content of GSK3beta and downregulated the remaining GSK3beta activity by Akt-dependent inhibition, consistent with minimal changes in phospho-T212. In summary, these results strongly suggest that in primary cultures, Akt selectively phosphorylates tau at S214 rather than T212. Our studies raise the possibility that tau S214 may participate in Akt-mediated anti-apoptotic signaling.

Possible roles of the complement (C) system in the normal and injured brain were explored with inbred mice that carried a frameshift mutation in the C5 gene. A congenic pair was used: the C5-sufficient (C5+) B10.D2/nSnJ strain with the functional allele (Hc1) from the C57BL/10J donor strain was compared with the C5-deficient (C5-) B10.D2/oSnJ with the Hc0 allele from the C5-deficient DBA/2J donor strain. In response to the excitotoxin kainic acid (KA), C5- mice had more hippocampal pyramidal neuron death and greater induction of astrocyte mRNAs (GFAP, apoE, apoJ). In primary astrocyte cultures from unlesioned mice, an inflammatory stimulus (LPS) caused greater production of IL-6 and TNF production in C5- mice. These enhanced responses to KA and LPS suggest that hereditary C5 deficits modify responses to neurodegenerative stimuli of neurons and astrocytes. Moreover, unlesioned C5- mice had smaller input-output slopes for the NMDA component of the EPSP amplitude, but enhanced the Ca(+2)-dependent AMPA binding. Thus, C5 deficits also modify basal properties of glutamatergic neurotransmission that pertain to synaptic plasticity. These findings are also discussed in relation to roles of the C-system in Alzheimer disease (AD). C5 deficiencies may also be considered in the choice of strains as transgene hosts and for genetic analysis of normal and pathological brain functions. In recent transgenic studies for AD, C5- hosts showed greater neurodegeneration, consistent with the present data. These pleiotropic associations of C5 deficiency indicate roles for the C-system in neurodegeneration, but also in normal neural functions.

This study characterizes the effect of transforming growth factor (TGF) beta 1 on clusterin expression in rat brain cells. 24 h after an acute unilateral intracerebroventricular infusion of TGF-beta 1, clusterin mRNA prevalence was increased in astrocytes that contained immunoreactive (IR) glial fibrillary acidic protein (GFAP). TGF-beta 1 selectively induced clusterin mRNA in astrocytes, as no clusterin mRNA was detected in neurons, oligodendrocytes, or microglia. TGF-beta 1 induced a bilateral increase in clusterin mRNA per astrocyte. Astrocyte hypertrophy (GFAP-IR area) was only increased on the ipsilateral side. In pure astrocyte cultures, TGF-beta 1 (200 pM) decreased clusterin mRNA levels and the rate of clusterin RNA transcription. However, in cultures of astrocytes that contained microglia and oligodendrocytes (mixed glia cultures), TGF-beta 1 caused a dose-dependent increase in astrocytic clusterin mRNA levels. The astrocytes that responded to TGF-beta 1 included two GFAP-IR subtypes, type 1 and 2. TGF-beta 1 increased clusterin protein in the conditioned medium from cultured glia, in either monotypic or mixed glial cultures. Thus, TGF-beta 1 and heterotypic cell interactions influence clusterin expression by astrocytes and may be important to the role of clusterin in multiple sclerosis, AIDS, and Alzheimer's disease.

This study concerns expression of the genes encoding three multifunctional proteins: clusterin and two complement cascade components, C1q and C4. Previous work from this and other laboratories has established that clusterin, Clq and C4 messenger RNAs are elevated during Alzheimer's disease, and in response to deafferenting and excitotoxic brain lesion. This study addresses hippocampal clusterin, ClqB and C4 expression in response to neurotoxins that caused selective neuron death. Kainate, which preferentially kills hippocampal CA3 pyramidal neurons but not dentate gyrus granule neurons induced clusterin immunoreactivity in CA1 and CA3 pyramidal neurons and adjacent astrocytes, but not in dentate gyrus granule neurons. In contrast, colchicine, which preferentially kills the dentate gyrus granule neurons, induced clusterin immunoreactivity in the local neuropil as punctate deposits, but not in the surviving or degenerating dentate gyrus granule neurons. Clusterin messenger RNA was increased in astrocytes. ClqB and C4 messenger RNAs increased within 48 h after kainate injections, particularly in the CA3 pyramidal layer, less in the dentate gyrus-CA4, and less in CA1. Clq immunoreactivity was detected in CA1 pyramidal neurons and also as small punctate deposits in the CA1 region at eight and 14 days after kainate. The increase of both clusterin and ClqB messenger RNAs after kainate injections was blocked by barbiturates that prevented seizures and neurodegeneration. In primary hippocampal neuronal cultures treated with glutamate, a subpopulation of cultured neurons that survived glutamate toxicity also had parallel elevations of clusterin and ClqB messenger RNA. In conclusion, cytotoxins that target selective hippocampal neurons increase the expression of both clusterin and ClqB in vivo and in vitro. These results show that elevations of clusterin messenger RNA or protein can be dissociated from each other and from cell death. These increased messenger RNAs were associated with immunoreactive deposits that differed by cell type and intra- versus extracellular locations. These results suggest that the complement system is involved in brain responses to injury.

This ultrastructural and light microscopic immunocytochemical study describes the time course of anatomical changes that occur in striatal astrocytes in response to neuronal deafferentation in young adult rats and the coordinate distribution of two astrocytic proteins involved in reactive synaptogenesis, glial fibrillary acidic protein and clusterin. We found that following a unilateral lesion of the cerebral cortex, striatal astrocytes undergo a rapid ultrastructural transformation from a protoplasmic to a reactive type of astroglia and are the primary cells involved in the removal of degenerating axon terminals, but not axons of passage, from the neuropil. In addition, at 10 and 27 days postlesion, processes of reactive astrocytes are also seen to occupy vacant postsynaptic spines after degenerating presynaptic terminals are removed, suggesting that they may also participate in the reinnervation of the deafferented neurons. By immunocytochemistry, reactive astrocytes were characterized by a significant increase in the intensity of glial fibrillary acidic protein staining beginning at three days postlesion and lasting for at least 27 days postlesion. Reactive astrocytes were characterized by cellular hypertrophy and an increase in the density of immunoreactive processes distributed throughout the deafferented striatum. However, our analysis of astrocyte cell number found no evidence of astrocyte proliferation in response to the deafferentation lesion. Although previous in situ hybridization studies have reported elevated clusterin messenger RNA in reactive astrocytes after decortication, clusterin immunoreactivity was not seen in the cell soma of reactive astrocytes but was distributed as punctate deposits, ranging from 1 to 2 microns in diameter, within the neuropil of the deafferented striatum. At 10 days postlesion, the distribution of clusterin staining appeared as large aggregates of immunoreactive deposits adjacent to neurons. However, by 27 days postlesion, the aggregates of clusterin reaction product were replaced by a fine scattering of individual punctate deposits distributed evenly over the dorsal part of the deafferented striatum. These data support the notion that reactive astrocytes serve multiple, time-dependent roles in response to brain injury and are involved in both the removal of degenerative debris from the lesion site as well as in reforming the synaptic circuitry of the damaged brain. Our data suggest that, in response to decortication, reactive astrocytes are the primary cells responsible for removing degenerating axon terminals, but not axons of passage, from the deafferented striatum and that the coordinate increase in glial fibrillary acidic protein may serve to stabilize the extension of reactive astrocytic processes during phagocytosis.(ABSTRACT TRUNCATED AT 400 WORDS)

Clusterin (SGP-2) is a newly described glycoprotein associated with several putative functions including responses to brain injury. This study reports the regional and cell type expression of clusterin mRNA and its encoded glycoprotein in the rat brain; a limited comparison was also done with the human brain. Using in situ hybridization combined with immunocytochemistry, we found that astrocytes and neurons may express clusterin mRNA in the normal adult brain. While astrocytes throughout the brain contained clusterin mRNA, there was regional selectivity for neuronal clusterin expression. In the striatum, clusterin mRNA was not detected in neurons. Only a subset of substantia nigra dopaminergic neurons or locus ceruleus noradrenergic neurons (tyrosine hydroxylase immunopositive) contained clusterin mRNA. However, neuronal clusterin mRNA was prevalent in pontine nuclei and in the red nucleus of the midbrain tegmentum. Similarly, clusterin mRNA was prevalent in both rat and human hippocampal neuron-specific enolase immunopositive pyramidal neurons, although rat CA1 neurons had less mRNA than CA2-CA3 neurons. Monotypic primary cell cultures from the neonatal rat showed clusterin mRNA in both neurons and astrocytes, but not in microglia. By immunocytochemistry, no clusterin immunopositive glia were observed in any region of the rat brain, confirming previous studies. However, clusterin immunopositive cells (putative neurons) were observed in the Purkinje cell layer of the cerebellum, medial and interposed cerebellar nuclei, trigeminal motor nucleus, and red nucleus. Finally, in vitro studies suggest that astrocytes, but not neurons, secrete clusterin, which is pertinent to clusterin immunodeposits found after experimental lesioning.

This investigation describes the schedule and regional distribution of astrocytic responses in striatum following deafferentation by unilateral frontal cortex ablation. In the ipsilateral deafferented striatum, glial fibrillary acidic protein and clusterin (sulfated glycoprotein-2) messengerRNA showed peak elevations by 10 days postlesioning (Northern blots). Vimentin messengerRNA responded faster, with a transient elevation by three days postlesioning. The messengerRNA for glial fibrillary acidic protein, clusterin and vimentin returned toward control levels by 27 days postlesioning. However, the neuronal marker growth-associated protein messengerRNA, was decreased at all postlesion times. By in situ hybridization, the increased glial fibrillary acidic protein messengerRNA and clusterin messengerRNA signals were localized mainly to the dorsal half of the ipsilateral deafferented striatum and followed the same schedule as found by Northern blots. Glial fibrillary acidic protein messengerRNA was widely diffused in the dorsal striatum and was excluded from fascicles of the internal capsule; a similar distribution was found for glial fibrillary acidic protein-immunopositive astrocytes. While clusterin messengerRNA signal showed a distinct clustering, its immunoreactivity appeared as deposits in the deafferented striatal neuropil; Western blots confirmed the immunocytochemical results. By in situ hybridization, vimentin messengerRNA was mostly localized to the cortical wound cavity dorsal to the deafferented striatum and overlapped the distribution of vimentin-immunopositive cells. These findings suggest a coordination of striatal astrocytic messengerRNA responses with the degeneration of corticostriatal afferents. We also compared these same parameters with those from published reports on the hippocampus after deafferenting lesions. Certain astrocyte molecular responses to deafferentation are detected about five days earlier in the hippocampus than in the striatum. This different schedule in response to decortication may pertain to differences in synaptic remodeling in the hippocampus vs striatum.

Unilateral ablation of the frontal cortex induced 30%-50% decrease of dopamine (DA) concentration in the ipsilateral striatum at 10 and 27 days after lesioning. There were increased ratios of dihydroxyphenylacetic acid (DOPAC): DA and homovanillic acid (HVA): DA by 20%-60% at 10 days post-lesioning, which suggest compensatory increases of DA metabolism. While no change in total striatal tyrosine hydroxylase (TH) polypeptide concentration was found at any post-lesion time, TH catalytic activity was decreased slightly (-25%) at 10 days. Among individual rats, at 3, 10 and 27 days post-lesioning, striatal DA concentration was inversely related to striatal glial fibrillary acidic protein (GFAP) concentration, a marker of astrocytic activity. The loss of DA was observed whether or not DA was normalized to striatal protein, which suggests that DA loss cannot be simply attributed to increased astrocytic proteins. These data suggest reciprocal relationships between the extent of astrocytic reactions after cortical deafferentation and striatal DA loss, which could involve local remodelling without primary damage to the nigro-striatal terminals.

A combination of in situ hybridization and immunocytochemistry (ICC) was used to identify the cells that contained mRNA for sulfated glycoprotein-2 (SGP-2) in adult male rats after striatal ibotenic acid (IA) lesioning. Astrocytic responses were monitored by ICC for glial fibrillary acidic protein (GFAP). IA lesioning that caused death of striatal neurons also stimulated astrocytic responses as monitored by GFAP and SGP-2. The SGP-2 immunoreactivity showed punctate deposits in the lesioned striatum without any apparent cellular localization. By in situ hybridization combined with ICC, SGP-2 mRNA was localized in astrocytes that were GFAP-immunopositive. These data suggest that reactive astrocytes may express SGP-2 which may be eventually secreted.