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Risk governance of nanomaterials and nanotechnologies has been traditionally mainly limited to risk assessment, risk management and life cycle assessment. Recent approaches have experimented with widening the scope and including economic, social, and ethical aspects. This paper reports on tests and stakeholder feedback on fine-tuning the use of ethical impact assessment guidelines (RiskGONE D3.6) and online tools adapting the CEN Workshop Agreement part 2 CWA 17145-2:2017 (E)) to support risk governance of nanomaterials, in the RiskGONE project. The EIA guidelines and tools are intended to be used as one module in a multicriteria decision support framework for risk governance of nanomaterials, but may also be used for a stand-alone ethical impact assessment.

Summary: Targeted inhibition of aberrant signaling is an important treatment strategy in cancer, but responses are often short-lived. Multi-drug combinations have the potential to mitigate this, but to avoid toxicity such combinations must be selective and given at low dosages. Here, we present a pipeline to identify promising multi-drug combinations. We perturbed an isogenic PI3K mutant and wild-type cell line pair with a limited set of drugs and recorded their signaling state and cell viability. We then reconstructed their signaling networks and mapped the signaling response to changes in cell viability. The resulting models, which allowed us to predict the effect of unseen combinations, indicated that no combination selectively reduces the viability of the PI3K mutant cells. However, we were able to validate 25 of the 30 combinations that we predicted to be anti-selective. Our pipeline enables efficient prioritization of multi-drug combinations from the enormous search space of possible combinations.

A drug used at the maximum tolerated dose can exert a strong selective pressure on cancer cells leading to resistance. In this study, the authors demonstrate the efficacy of using low dose of multiple drugs for preventing and treating resistance to EGFR tyrosine kinase inhibitors in NSCLC cells.

An amendment to this paper has been published and can be accessed via the original article.

Intrinsic and acquired resistances are major hurdles preventing the effective use of MEK inhibitors for treatment of colorectal cancer (CRC). Some 35–45% of colorectal cancers are KRAS-mutant and their treatment remains challenging as these cancers are refractory to MEK inhibitor treatment, because of feedback activation of receptor tyrosine kinases (RTKs). We reported previously that loss of ERN1 sensitizes a subset of KRAS-mutant colon cancer cells to MEK inhibition. Here we show that the loss of RUNX2 or its cofactor CBFB can confer MEK inhibitor resistance in CRC cells. Mechanistically, we find that cells with genetically ablated RUNX2 or CBFB activate multiple RTKs, which coincides with high SHP2 phosphatase activity, a phosphatase that relays signals from the cell membrane to downstream pathways governing growth and proliferation. Moreover, we show that high activity of SHP2 is causal to loss of RUNX2-induced MEK inhibitor resistance, as a small molecule SHP2 inhibitor reinstates sensitivity to MEK inhibitor in RUNX2 knockout cells. Our results reveal an unexpected role for loss of RUNX2/CBFB in regulating RTK activity in colon cancer, resulting in reduced sensitivity to MEK inhibitors.

Abstract Background Mutations in KRAS are frequent in human cancer, yet effective targeted therapeutics for these cancers are still lacking. Attempts to drug the MEK kinases downstream of KRAS have had limited success in clinical trials. Understanding the specific genomic vulnerabilities of KRAS-driven cancers may uncover novel patient-tailored treatment options. Methods We first searched for synthetic lethal (SL) genetic interactions with mutant RAS in yeast with the ultimate aim to identify novel cancer-specific targets for therapy. Our method used selective ploidy ablation, which enables replication of cancer-specific gene expression changes in the yeast gene disruption library. Second, we used a genome-wide CRISPR/Cas9-based genetic screen in KRAS mutant human colon cancer cells to understand the mechanistic connection between the synthetic lethal interaction discovered in yeast and downstream RAS signaling in human cells. Results We identify loss of the endoplasmic reticulum (ER) stress sensor IRE1 as synthetic lethal with activated RAS mutants in yeast. In KRAS mutant colorectal cancer cell lines, genetic ablation of the human ortholog of IRE1, ERN1, does not affect growth but sensitizes to MEK inhibition. However, an ERN1 kinase inhibitor failed to show synergy with MEK inhibition, suggesting that a non-kinase function of ERN1 confers MEK inhibitor resistance. To investigate how ERN1 modulates MEK inhibitor responses, we performed genetic screens in ERN1 knockout KRAS mutant colon cancer cells to identify genes whose inactivation confers resistance to MEK inhibition. This genetic screen identified multiple negative regulators of JUN N-terminal kinase (JNK) /JUN signaling. Consistently, compounds targeting JNK/MAPK8 or TAK1/MAP3K7, which relay signals from ERN1 to JUN, display synergy with MEK inhibition. Conclusions We identify the ERN1-JNK-JUN pathway as a novel regulator of MEK inhibitor response in KRAS mutant colon cancer. The notion that multiple signaling pathways can activate JUN may explain why KRAS mutant tumor cells are traditionally seen as highly refractory to MEK inhibitor therapy. Our findings emphasize the need for the development of new therapeutics targeting JUN activating kinases, TAK1 and JNK, to sensitize KRAS mutant cancer cells to MEK inhibitors.

Decision-making balances the benefits of additional information with the cost of time, but it is unclear whether humans adjust this balance within individual decisions. Here, authors show that we do make such adjustments to suit contextual demands and suggest that these are driven by modulation of neural gain.

Nature Communications 7: Article number: 13526 (2016); Published: 24 November 2016; Updated: 18 January 2017 In Fig. 4c of this Article, the colour bars were inadvertently changed from graded to solid during the production process. The correct version of Fig. 4c appears below as Fig. 1.

Glioblastoma cells rely on connexin 43 (Cx43)-based gap junctions (GJs) for intercellular communication, enabling them to integrate into a widely branched malignant network. Although there are promising prospects for new targeted therapies, the lack of clinically feasible GJ inhibitors has impeded their adoption in clinical practice. In the present study, we investigated tonabersat (TO), a blood-brain-barrier-penetrating drug with GJ-inhibitory properties, in regard to its potential to disassemble intercellular connectivity in glioblastoma networks. Fluorescence-guided measurements of calcein cell-to-cell transfer were used to study functional intercellular connectivity. Specific DNA fragmentation rates of propidium iodide-stained nuclei were measured as a surrogate readout for cell death using flow cytometry. CRISPR/Cas9-mediated gene editing of Cx43 served as a validation tool of cellular effects related to Cx43 GJ inhibition. 3' mRNA sequencing was performed for molecular downstream analysis. We found that TO reduced intercellular GJ-mediated cytosolic traffic and yielded a significant reduction of tumor microtube (TM) length. TO-mediated inhibition of cellular tumor networks was accompanied by a synergistic effect for temozolomide-induced cell death. CRISPR/Cas9 Cx43-knockout revealed similar results, indicating that TO-mediated inhibitory effects rely on the inhibition of Cx43-based GJs. Gene set enrichment analyses found that GJ-mediated synergistic cytotoxic effects were linked to a significant upregulation of cell death signaling pathways. In conclusion, TO disrupts TM-based network connectivity via GJ inhibition and renders glioblastoma cells more susceptible to cytotoxic therapy. Given its previous use in clinical trials for migraine therapy, TO might harbor the potential of bridging the idea of a GJ-targeted therapeutic approach from bench to bedside., (© 2024 The Author(s). Molecular Oncology published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Mounting evidence suggests a prominent role for alpha-synuclein (a-syn) in neuronal cell function. Alterations in the levels of cellular a-syn have been hypothesized to play a critical role in the development of Parkinson's disease (PD); however, mechanisms that control expression of the gene for a-syn (SNCA) in cis and trans as well as turnover of a-syn are not well understood. We analyzed whether methyl-CpG binding protein 2 (MeCP2), a protein that specifically binds methylated DNA, thus regulating transcription, binds at predicted binding sites in intron 1 of the SNCA gene and regulates a-syn protein expression. Chromatin immunoprecipitation (ChIP) and electrophoretic mobility-shift assays (EMSA) were used to confirm binding of MeCP2 to regulatory regions of SNCA. Site-specific methylation and introduction of localized mutations by CRISPR/Cas9 were used to investigate the binding properties of MeCP2 in human SK-N-SH neuroblastoma cells. The significance of MeCP2 for SNCA regulation was further investigated by overexpressing MeCP2 and mutated variants of MeCP2 in MeCP2 knockout cells. We found that methylation-dependent binding of MeCP2 at a restricted region of intron 1 of SNCA had a significant impact on the production of a-syn. A single nucleotide substitution near to CpG1 strongly increased the binding of MeCP2 to intron 1 of SNCA and decreased a-syn protein expression by 60%. In contrast, deletion of a single nucleotide closed to CpG2 led to reduced binding of MeCP2 and significantly increased a-syn levels. In accordance, knockout of MeCP2 in SK-N-SH cells resulted in a significant increase in a-syn production, demonstrating that SNCA is a genomic target for MeCP2 regulation. In addition, the expression of two mutated MeCP2 variants found in Rett syndrome (RTT) showed a loss of their ability to reduce a-syn expression. This study demonstrates that methylation of CpGs and binding of MeCP2 to intron 1 of the SNCA gene plays an important role in the control of a-syn expression. In addition, the changes in SNCA regulation found by expression of MeCP2 variants carrying mutations found in RTT patients may be of importance for the elucidation of a new molecular pathway in RTT, a rare neurological disorder caused by mutations in MECP2., (© 2024. The Author(s).)

The spinocerebellar ataxias (SCA) comprise a group of inherited neurodegenerative diseases. Machado-Joseph Disease (MJD) or spinocerebellar ataxia 3 (SCA3) is the most common autosomal dominant form, caused by the expansion of CAG repeats within the ataxin-3 (ATXN3) gene. This mutation results in the expression of an abnormal protein containing long polyglutamine (polyQ) stretches that confers a toxic gain of function and leads to misfolding and aggregation of ATXN3 in neurons. As a result of the neurodegenerative process, SCA3 patients are severely disabled and die prematurely. Several screening approaches, e.g., druggable genome-wide and drug library screenings have been performed, focussing on the reduction in stably overexpressed ATXN3(polyQ) protein and improvement in the resultant toxicity. Transgenic overexpression models of toxic ATXN3, however, missed potential modulators of endogenous ATXN3 regulation. In another approach to identify modifiers of endogenous ATXN3 expression using a CRISPR/Cas9-modified SK-N-SH wild-type cell line with a GFP - T2A - luciferase ( LUC ) cassette under the control of the endogenous ATXN3 promotor, four statins were identified as potential activators of expression. We here provide an overview of the high throughput screening approaches yet performed to find compounds or genomic modifiers of ATXN3(polyQ) toxicity in different SCA3 model organisms and cell lines to ameliorate and halt SCA3 progression in patients. Furthermore, the putative role of cholesterol in neurodegenerative diseases (NDDs) in general and SCA3 in particular is discussed.

Glioblastoma is the most common and aggressive primary tumor of the central nervous system with poor outcome. Current gold standard treatment is surgical resection followed by a combination of radio- and chemotherapy. Efficacy of temozolomide (TMZ), the primary chemotherapeutic agent, depends on the DNA methylation status of the O6-methylguanine DNA methyltransferase (MGMT), which has been identified as a prognostic biomarker in glioblastoma patients. Clinical studies revealed that glioblastoma patients with hypermethylated MGMT promoter have a better response to TMZ treatment and a significantly improved overall survival. In this study, we thus used the CRISPRoff genome editing tool to mediate targeted DNA methylation within the MGMT promoter region. The system carrying a CRISPR-deactivated Cas9 (dCas9) fused with a methyltransferase (Dnmt3A/3L) domain downregulated MGMT expression in TMZ-resistant human glioblastoma cell lines through targeted DNA methylation. The reduction of MGMT expression levels reversed TMZ resistance in TMZ-resistant glioblastoma cell lines resulting in TMZ induced dose-dependent cell death rates. In conclusion, we demonstrate targeted RNA-guided methylation of the MGMT promoter as a promising tool to overcome chemoresistance and improve the cytotoxic effect of TMZ in glioblastoma., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Inc.)

The deubiquitinating enzyme Ataxin-3 (ATXN3) contains a polyglutamine (PolyQ) region, the expansion of which causes spinocerebellar ataxia type-3 (SCA3). ATXN3 has multiple functions, such as regulating transcription or controlling genomic stability after DNA damage. Here we report the role of ATXN3 in chromatin organization during unperturbed conditions, in a catalytic-independent manner. The lack of ATXN3 leads to abnormalities in nuclear and nucleolar morphology, alters DNA replication timing and increases transcription. Additionally, indicators of more open chromatin, such as increased mobility of histone H1, changes in epigenetic marks and higher sensitivity to micrococcal nuclease digestion were detected in the absence of ATXN3. Interestingly, the effects observed in cells lacking ATXN3 are epistatic to the inhibition or lack of the histone deacetylase 3 (HDAC3), an interaction partner of ATXN3. The absence of ATXN3 decreases the recruitment of endogenous HDAC3 to the chromatin, as well as the HDAC3 nuclear/cytoplasm ratio after HDAC3 overexpression, suggesting that ATXN3 controls the subcellular localization of HDAC3. Importantly, the overexpression of a PolyQ-expanded version of ATXN3 behaves as a null mutant, altering DNA replication parameters, epigenetic marks and the subcellular distribution of HDAC3, giving new insights into the molecular basis of the disease., (© The Author(s) 2023. Published by Oxford University Press on behalf of Nucleic Acids Research.)

Background: Glioblastoma cells assemble to a syncytial communicating network based on tumor microtubes (TMs) as ultra-long membrane protrusions. The relationship between network architecture and transcriptional profile remains poorly investigated. Drugs that interfere with this syncytial connectivity such as meclofenamate (MFA) may be highly attractive for glioblastoma therapy., Methods: In a human neocortical slice model using glioblastoma cell populations of different transcriptional signatures, three-dimensional tumor networks were reconstructed, and TM-based intercellular connectivity was mapped on the basis of two-photon imaging data. MFA was used to modulate morphological and functional connectivity; downstream effects of MFA treatment were investigated by RNA sequencing and fluorescence-activated cell sorting (FACS) analysis., Results: TM-based network morphology strongly differed between the transcriptional cellular subtypes of glioblastoma and was dependent on axon guidance molecule expression. MFA revealed both a functional and morphological demolishment of glioblastoma network architectures which was reflected by a reduction of TM-mediated intercellular cytosolic traffic as well as a breakdown of TM length. RNA sequencing confirmed a downregulation of NCAM and axon guidance molecule signaling upon MFA treatment. Loss of glioblastoma communicating networks was accompanied by a failure in the upregulation of genes that are required for DNA repair in response to temozolomide (TMZ) treatment and culminated in profound treatment response to TMZ-mediated toxicity., Conclusion: The capacity of TM formation reflects transcriptional cellular heterogeneity. MFA effectively demolishes functional and morphological TM-based syncytial network architectures. These findings might pave the way to a clinical implementation of MFA as a TM-targeted therapeutic approach., (© The Author(s) 2021. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Multiplications, mutations and dysregulation of the alpha synuclein gene (SNCA) are associated with the demise of dopaminergic neurons and are considered to play important roles in the pathogenesis of familial and sporadic forms of Parkinson's disease. Regulation of SNCA expression might thus be an appropriate target for treatment. We aimed to identify specific modulators of SNCA transcription, generated CRISPR/Cas9 modified SNCA-GFP-luciferase (LUC) genomic fusion- and control cell lines and screened a library of 1649 bioactive compounds, including the FDA approved drugs. We found no inhibitors but three selective activators which increased SNCA mRNA and protein levels., (© 2021. The Author(s).)

Glioblastoma is a malignant brain tumor and one of the most lethal cancers in human. Temozolomide constitutes the standard chemotherapeutic agent, but only shows limited efficacy in glioblastoma patients with unmethylated O-6-methylguanine-DNA methyltransferase (MGMT) promoter status. Recently, it has been shown that glioblastoma cells communicate via particular ion-channels-so-called gap junctions. Interestingly, inhibition of these ion channels has been reported to render MGMT promoter-methylated glioblastoma cells more susceptible for a therapy with temozolomide. However, given the percentage of about 65% of glioblastoma patients with an unmethylated MGMT promoter methylation status, this treatment strategy is limited to only a minority of glioblastoma patients. In the present study we show that-in contrast to temozolomide-pharmacological inhibition of intercellular cytosolic traffic via gap junctions reinforces the antitumoral effects of chemotherapeutic agent lomustine, independent of MGMT promoter methylation status. In view of the growing interest of lomustine in glioblastoma first and second line therapy, these findings might provide a clinically-feasible way to profoundly augment chemotherapeutic effects for all glioblastoma patients.

Gap junctions have recently been shown to interconnect glioblastoma cells to a multicellular syncytial network, thereby allowing intercellular communication over long distances as well as enabling glioblastoma cells to form routes for brain microinvasion. Against this backdrop gap junction-targeted therapies might provide for an essential contribution to isolate cancer cells within the brain, thus increasing the tumor cells' vulnerability to the standard chemotherapeutic agent temozolomide. By utilizing INI-0602-a novel gap junction inhibitor optimized for crossing the blood brain barrier-in an oncological setting, the present study was aimed at evaluating the potential of gap junction-targeted therapy on primary human glioblastoma cell populations. Pharmacological inhibition of gap junctions profoundly sensitized primary glioblastoma cells to temozolomide-mediated cell death. On the molecular level, gap junction inhibition was associated with elevated activity of the JNK signaling pathway. With the use of a novel gap junction inhibitor capable of crossing the blood-brain barrier-thus constituting an auspicious drug for clinical applicability-these results may constitute a promising new therapeutic strategy in the field of current translational glioblastoma research.

More than 90% of the human genome are transcribed as non-coding RNAs. While it is still under debate if all these non-coding transcripts are functional, there is emerging evidence that RNA has several important functions in addition to coding for proteins. For example, microRNAs (miRNAs) are important regulatory RNAs that control gene expression in various biological processes and human diseases. In spinocerebellar ataxia type 3 (SCA3), a devastating neurodegenerative disease, miRNAs are involved in the disease process at different levels, including the deregulation of components of the general miRNA biogenesis machinery, as well as in the cell type-specific control of the expression of the SCA3 disease protein and other SCA3 disease-relevant proteins. However, it remains difficult to predict whether these changes are a cause or a consequence of the neurodegenerative process in SCA3. Further studies using standardized procedures for the analysis of miRNA expression and larger sample numbers are required to enhance our understanding of the miRNA-mediated processes involved in SCA3 disease and may enable the development of miRNA-based therapeutics. In this review, we summarize the findings of independent studies highlighting both the disease-related and cytoprotective roles of miRNAs that have been implicated so far in the disease process of SCA3., (Copyright © 2019. Published by Elsevier Ltd.)

Background: Spinocerebellar ataxia type 3 (SCA3), the most common spinocerebellar ataxia, is caused by a polyglutamine (polyQ) expansion in the protein ataxin-3 (ATXN3). Silencing the expression of polyQ-expanded ATXN3 rescues the cellular disease phenotype., Objective: This study investigated the differential expression of microRNAs (miRNAs), small noncoding RNAs targeting gene expression, in lymphoblastoid cells (LCs) from SCA3 patients and the capability of identified deregulated miRNAs to target and alter ATXN3 expression., Methods: MiRNA profiling was performed by microarray hybridization of total RNA from control and SCA3-LCs. The capability of the identified miRNAs and their target sites to suppress ATXN3 expression was analyzed using mutagenesis, reverse transcription PCR, immunoblotting, luciferase reporter assays, mimics and precursors of the identified miRNAs., Results: SCA3-LCs showed significantly decreased expression levels of ATXN3 and a significant upregulation of the ATXN3-3'UTR targeting miRNAs, miR-32 and miR-181c and closely related members of the miR-25 and miR-181 family, respectively. MiR-32 and miR-181c effectively targeted the 3'UTR of ATXN3 and suppressed the expression of ATXN3., Conclusions: The simultaneous upregulation of closely related miRNAs targeting the 3'UTR of ATXN3 and the significantly reduced ATXN3 expression levels in SCA3-LCs suggests that miR-25 and miR-181 family members cooperatively bind to the 3'UTR to suppress the expression of ATXN3. The findings further suggest that the upregulation of miR-25 and miR-181 family members in SCA3- LCs reflects a cell type-specific, protective mechanism to diminish polyQ-mediated cytotoxic effects. Thus, miRNA mimics of miR-25 and miR-181 family members may prove useful for the treatment of SCA3., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.)

Molecular chaperones are important regulators of protein folding and proteasomal removal of misfolded proteins. In spinocerebellar ataxia type 3 (SCA3), the co-chaperone DnaJ homology subfamily B member 1 (DNAJB1 or heat shock protein 40) is recruited to protein aggregates formed by the disease-causing mutant polyglutamine (polyQ) protein ataxin-3 (ATXN3). Over-expression of DNAJB1 reduces polyQ protein toxicity. Here, we identified two miRNAs, miR-370 and miR-543, that function in posttranscriptional regulation of DNAJB1 expression. MiRNAs are small endogenously produced RNAs controlling mRNA stability and play a role in polyQ disease pathogenesis. In human neuronal cultures derived from SCA3 patient-specific induced pluripotent stem cell (iPSC) lines, miR-370 and miR-543 levels are upregulated, while DNAJB1 expression is concurrently reduced. These findings suggest that downregulation of DNAJB1 by these two miRNAs is an early event that could contribute to SCA3 pathogenesis. Inhibition of these two miRNAs in turn could stabilize DNAJB1 and thereby be beneficial in SCA3 disease., Competing Interests: The authors have declared that no competing interests exist.

Guillain-Barré syndrome (GBS) is an acute, immune-mediated polyradiculoneuropathy characterized by rapidly progressive paresis and sensory disturbances. Moderate to severe and often intractable neuropathic pain is a common symptom of GBS, but its underlying mechanisms are unknown. Pathology of GBS is classically attributed to demyelination of large, myelinated peripheral fibers. However, there is increasing evidence that neuropathic pain in GBS is associated with impaired function of small, unmyelinated, nociceptive fibers. We therefore examined the functional properties of small DRG neurons, the somata of nociceptive fibers, in a rat model of GBS (experimental autoimmune neuritis=EAN). EAN rats developed behavioral signs of neuropathic pain. This was accompanied by a significant shortening of action potentials due to a more rapid repolarization and an increase in repetitive firing in a subgroup of capsaicin-responsive DRG neurons. Na + current measurements revealed a significant increase of the fast TTX-sensitive current and a reduction of the persistent TTX-sensitive current component. These changes of Na + currents may account for the significant decrease in AP duration leading to an overall increase in excitability and are therefore possibly directly linked to pathological pain behavior. Thus, like in other animal models of neuropathic and inflammatory pain, Na + channels seem to be crucially involved in the pathology of GBS and may constitute promising targets for pain modulating pharmaceuticals., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Tight regulation of the balance between self-renewal and differentiation of neural stem cells is crucial to assure proper neural development. In this context, Notch signaling is a well-known promoter of stemness. In contrast, the bifunctional brain-enriched microRNA miR-9/9(∗) has been implicated in promoting neuronal differentiation. Therefore, we set out to explore the role of both regulators in human neural stem cells. We found that miR-9/9(∗) decreases Notch activity by targeting NOTCH2 and HES1, resulting in an enhanced differentiation. Vice versa, expression levels of miR-9/9(∗) depend on the activation status of Notch signaling. While Notch inhibits differentiation of neural stem cells, it also induces miR-9/9(∗) via recruitment of the Notch intracellular domain (NICD)/RBPj transcriptional complex to the miR-9/9(∗)_2 genomic locus. Thus, our data reveal a mutual interaction between bifunctional miR-9/9(∗) and the Notch signaling cascade, calibrating the delicate balance between self-renewal and differentiation of human neural stem cells., (Copyright © 2016 The Author(s). Published by Elsevier Inc. All rights reserved.)

Machado-Joseph disease (MJD; also called spinocerebellar ataxia type 3) is a dominantly inherited late-onset neurodegenerative disorder caused by expansion of polyglutamine (polyQ)-encoding CAG repeats in the MJD1 gene (also known as ATXN3). Proteolytic liberation of highly aggregation-prone polyQ fragments from the protective sequence of the MJD1 gene product ataxin 3 (ATXN3) has been proposed to trigger the formation of ATXN3-containing aggregates, the neuropathological hallmark of MJD. ATXN3 fragments are detected in brain tissue of MJD patients and transgenic mice expressing mutant human ATXN3(Q71), and their amount increases with disease severity, supporting a relationship between ATXN3 processing and disease progression. The formation of early aggregation intermediates is thought to have a critical role in disease initiation, but the precise pathogenic mechanism operating in MJD has remained elusive. Here we show that L-glutamate-induced excitation of patient-specific induced pluripotent stem cell (iPSC)-derived neurons initiates Ca(2+)-dependent proteolysis of ATXN3 followed by the formation of SDS-insoluble aggregates. This phenotype could be abolished by calpain inhibition, confirming a key role of this protease in ATXN3 aggregation. Aggregate formation was further dependent on functional Na(+) and K(+) channels as well as ionotropic and voltage-gated Ca(2+) channels, and was not observed in iPSCs, fibroblasts or glia, thereby providing an explanation for the neuron-specific phenotype of this disease. Our data illustrate that iPSCs enable the study of aberrant protein processing associated with late-onset neurodegenerative disorders in patient-specific neurons.

Ataxin-3 (ATXN3), the disease protein in spinocerebellar ataxia type 3 (SCA3), binds to target gene promoters and modulates transcription by interaction with transcriptional regulators. Here, we show that ATXN3 interacts with the forkhead box O (FOXO) transcription factor FOXO4 and activates the FOXO4-dependent transcription of the manganese superoxide dismutase (SOD2) gene. Upon oxidative stress, ATXN3 and FOXO4 translocate to the nucleus, concomitantly bind to the SOD2 gene promoter and increase the expression of the antioxidant enzyme SOD2. Compared with normal ATXN3, mutant ATXN3 has a reduced capability to activate the FOXO4-mediated SOD2 expression and interferes with binding of FOXO4 to the SOD2 gene promoter. These findings are consistent with a downregulation of SOD2 in pontine brain tissue and lymphoblastoid cell (LC) lines of SCA3 patients. In response to oxidative stress, LCs from SCA3 patients show a specific impairment to upregulate SOD2 expression in correlation with a significantly increased formation of reactive oxygen species and cytotoxicity. The impairment to increase the expression of SOD2 under oxidative stress conditions is associated with a significantly reduced binding of FOXO4 to the SOD2 gene promoter in SCA3-LCs. Finally and consistent with a regulatory role of ATXN3 in SOD2 expression, knockdown of endogenous ATXN3 by RNA interference represses the expression of SOD2. These findings support that ATXN3 plays an important role in regulating the FOXO4-dependent antioxidant stress response via SOD2 and suggest that a decreased antioxidative capacity and increased susceptibility towards oxidative stress contributes to neuronal cell death in SCA3.

Expansion of a polymorphic polyglutamine segment is the common denominator of neurodegenerative polyglutamine diseases. The expanded proteins typically accumulate in large intranuclear inclusions and induce neurodegeneration. However, the mechanisms that determine the subcellular site and rate of inclusion formation are largely unknown. We found that the conserved putative nuclear localization sequence Arg-Lys-Arg-Arg, which is retained in a highly aggregation-prone fragment of ataxin-3, did not affect the site and degree of inclusion formation in a cell culture model of spinocerebellar ataxia type 3. Addition of synthetic nuclear export or import signals led to the expected localization of ataxin-3 and determined the subcellular site of aggregate formation. Triggering a cellular stress response by heat shock transcription factor DeltaHSF1 coexpression abrogated aggregation in the cytoplasm but not in the nucleus. These findings indicate that native aggregation-prone fragments derived from expanded ataxin-3 may eventually escape the cytoplasmic quality control, resulting in aggregation in the nuclear compartment.

The nuclear presence of the expanded disease proteins is of critical importance for the pathogeneses of polyglutamine diseases. Here we show that protein casein kinase 2 (CK2)-dependent phosphorylation controls the nuclear localization, aggregation and stability of ataxin-3 (ATXN3), the disease protein in spinocerebellar ataxia type 3 (SCA3). Serine 340 and 352 within the third ubiquitin-interacting motif of ATXN3 were particularly important for nuclear localization of normal and expanded ATXN3 and mutation of these sites robustly reduced the formation of nuclear inclusions; a putative nuclear leader sequence was not required. ATXN3 associated with CK2alpha and pharmacological inhibition of CK2 decreased nuclear ATXN3 levels and the formation of nuclear inclusions. Moreover, we found that ATXN3 shifted to the nucleus upon thermal stress in a CK2-dependent manner, indicating a key role of CK2-mediated phosphorylation of ATXN3 in SCA3 pathophysiology.

Increasing evidence has linked inflammatory processes to neurodegenerative disorders, including Alzheimer's and Parkinson's disease (PD). Tumor necrosis factor alpha (TNF-alpha) is a key inflammatory cytokine and several studies linked increased TNF-alpha to dopaminergic cell death in PD. The TNF-alpha promoter sequence contains several CpG dinucleotides located within or next to transcription factor binding sites. To test the hypothesis whether the methylation state of the TNF-alpha promoter contributes to increased expression of TNF-alpha in PD we compared DNA from different brain regions (substantia nigra pars compacta (SNpc) and cortex) of PD patients and neurologically healthy, age and sex matched controls by bisulfite sequencing of the TNF-alpha promoter region. The TNF-alpha promoter DNA from SNpc was significantly less methylated in comparison to DNA from cortex; however both in PD patients and controls. Although there was a tendency for hypomethylation in PD, our analysis of the 10 CpGs in the TNF-alpha core promoter region (-258 to -35 relative to the TSS) revealed no particular pattern in PD patients compared to control and identified no particular hypomethylated position in cortex or SNpc DNA. Electrophoretic mobility shift and luciferase reporter assays showed that methylation of specific solitary CpG in the TNF-alpha promoter resulted in reduced binding of the transcription factors AP-2 and Sp1, respectively, and suppressed TNF-alpha promoter activity. The brain region specific methylation state of solitary CpG in the TNF-alpha promoter thus determines transcription factor binding efficacy and TNF-alpha expression. A lesser degree of methylation of the TNF-alpha promoter in SNpc cells could underlie the increased susceptibility of dopaminergic neurons to TNF-alpha mediated inflammatory reactions.

Spinocerebellar ataxia type 3 is a neurodegenerative disease caused by expansion of a polyglutamine domain in the protein ataxin-3 (ATXN3). Physiological functions of ATXN3 presumably include ubiquitin protease and transcriptional corepressor activity. To gain insight into the function of ATXN3 and to test the hypothesis that loss of ATXN3 contributes to the pathology in SCA3 we generated Atxn3 knockout (ko) mice by targeted mutagenesis. Loss of Atxn3 did not affect viability or fertility and Atxn3 ko mice displayed no overt abnormalities. On the accelerating Rotarod Atxn3 ko mice performed as well as wildtype (wt) animals, but reduced exploratory behavior in the open field suggested a sense of heightened anxiety. While no gross deficits were apparent upon morphological examination, we found increased levels of ubiquitinated proteins in Atxn3 ko tissues. Thus Atxn3 ko mice provide the first in vivo reference to the deubiquitinating activity of ATXN3.

Mutations in the valosin-containing protein (VCP, p97) gene on chromosome 9p13-p12 cause a late-onset form of autosomal dominant inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia (IBMPFD). We report on the pathological consequences of three heterozygous VCP (R93C, R155H, R155C) mutations on human striated muscle. IBMPFD skeletal muscle pathology is characterized by degenerative changes and filamentous VCP- and ubiquitin-positive cytoplasmic and nuclear protein aggregates. Furthermore, this is the first report demonstrating that mutant VCP leads to a novel form of dilatative cardiomyopathy with inclusion bodies. In contrast to post-mitotic striated muscle cells and neurons of IBMPFD patients, evidence of protein aggregate pathology was not detected in primary IBMPFD myoblasts or in transient and stable transfected cells using wild-type-VCP and R93C-, R155H-, R155C-VCP mutants. Glutathione S-transferase pull-down experiments showed that all three VCP mutations do not affect the binding to Ufd1, Npl4 and ataxin-3. Structural analysis demonstrated that R93 and R155 are both surface-accessible residues located in the centre of cavities that may enable ligand-binding. Mutations at R93 and R155 are predicted to induce changes in the tertiary structure of the VCP protein. The search for putative ligands to the R93 and R155 cavities resulted in the identification of cyclic sugar compounds with high binding scores. The latter findings provide a novel link to VCP carbohydrate interactions in the complex pathology of IBMPFD.

Ataxin-3 (AT3), the disease protein in spinocerebellar ataxia type 3 (SCA3), has been associated with the ubiquitin-proteasome system and transcriptional regulation. Here we report that normal AT3 binds to target DNA sequences in specific chromatin regions of the matrix metalloproteinase-2 (MMP-2) gene promoter and represses transcription by recruitment of the histone deacetylase 3 (HDAC3), the nuclear receptor corepressor (NCoR), and deacetylation of histones bound to the promoter. Both normal and expanded AT3 physiologically interacted with HDAC3 and NCoR in a SCA3 cell model and human pons tissue; however, normal AT3-containing protein complexes showed increased histone deacetylase activity, whereas expanded AT3-containing complexes had reduced deacetylase activity. Consistently, histone analyses revealed an increased acetylation of total histone H3 in expanded AT3-expressing cells and human SCA3 pons. Expanded AT3 lost the repressor function and displayed altered DNA/chromatin binding that was not associated with recruitment of HDAC3, NCoR, and deacetylation of the promoter, allowing aberrant MMP-2 transcription via the transcription factor GATA-2. For transcriptional repression normal AT3 cooperates with HDAC3 and requires its intact ubiquitin-interacting motifs (UIMs), whereas aberrant transcriptional activation by expanded AT3 is independent of the UIMs but requires the catalytic cysteine of the ubiquitin protease domain. These findings demonstrate that normal AT3 binds target promoter regions and represses transcription of a GATA-2-dependent target gene via formation of histone-deacetylating repressor complexes requiring its UIM-associated function. Expanded AT3 aberrantly activates transcription via its catalytic site and loses the ability to form deacetylating repressor complexes on target chromatin regions.

Objective: We analyzed the expression of the inflammatory mediators IL-1beta, IL-1ra, IL-6 and the transcription factors IRF-1 and C/EBPdelta (previously identified in a transgenic model of spinocerebellar ataxia type 3 (SCA3) by gene expression profiling) in the central nervous system of SCA3 patients in relation to neuronal cell loss and ataxin-3-positive neuronal intranuclear inclusions (NI), to identify a putative upregulation of cytokines or microglia in SCA3 brains and to investigate whether enhanced cytokine expression was a generalized event mediating neuronal dysfunction in SCA3., Materials and Methods: Light- and electronmicroscopic immunohistochemistry was performed on SCA3 tissues derived from five patients from unrelated families with genetically confirmed diagnosis, and six individuals without a history of neurological or inflammatory disease., Results: NI were found almost exclusively in brain regions that also showed neuronal cell loss, i.e. in pons and dentate nucleus neurons, rarely in putamen and thalamus, but not in cerebral or cerebellar cortex. NI displayed an irregular surface and were mostly attached to the nucleoli. Quantitative analysis of NI in the pons revealed an inverse relation of NI and cell loss, i.e. patients with more severe neuronal cell loss had a smaller proportion of neurons with NI. Thus, formation of NI is not necessarily an indicator of cell death but could exert a protective effect. We found increased expression of IL-1beta, IL-1ra, IL-6 and C/EBPdelta only in pons and dentate nucleus neurons and both in neurons with and without NI, suggesting that NI are not a prerequisite for transcriptional changes., Conclusions: Our data suggest that the selectively affected neuronal populations in SCA3 undergo a complex alteration of gene expression independent from the formation of NI.

Children with hereditary severe hyperhomocysteinemia present with a variety of neurological impairment, and mild hyperhomocysteinemia has been associated with neurodegeneration in the elderly. The link of hyperhomocysteinemia to neurological dysfunction is unknown. We investigated mitochondrial mechanisms of homocysteine (HCys) neurotoxicity in rat dopaminergic pheochromocytoma cells, human neuroblastoma cells and primary rat cerebellar granule neurons. HCys dose dependently impaired cytochrome c oxidase (COX) activity as well as stability and induced reactive oxygen species and apoptotic cell death. We found that HCys binds the COX cofactor Cu(2+), and Cu(2+) supplementation prior to HCys treatment preserved COX activity and prevented cell death. The Cu(2+) chelating action of HCys and impairement of COX activity represent novel mechanisms of HCys neurotoxicity, which might be preventable by supplementation of Cu(2+).

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant inherited neurodegenerative disease caused by the expansion of a polyglutamine repeat within the disease protein, ataxin-3. There is growing evidence that neuronal electrophysiological properties are altered in a variety of polyglutamine diseases such as Huntington's disease and SCA1 and that these alterations may contribute to disturbances of neuronal function prior to neurodegeneration. To elucidate possible electrophysiological changes in SCA3, we generated a stable PC12 cell model with inducible expression of normal and mutant human full-length ataxin-3 and analyzed the electrophysiological properties after induction of the recombinant ataxin-3 expression. Neuronally differentiated PC12 cells expressing the expanded form of ataxin-3 showed significantly decreased viabilities and developed ultrastructural changes resembling human SCA3. Prior to neuronal cell death, we found a significant reduction of the resting membrane potential and a hyperpolarizing shift of the activation curve of the delayed rectifier potassium current. These findings indicate that electrophysiological properties are altered in mutant ataxin-3 expressing neuronal cells and may contribute to neuronal dysfunction in SCA3.

Multiple system atrophy (MSA) and sporadic, non-mendelian Parkinson's disease (PD) are progressive neurodegenerative disorders with overlapping clinical symptoms and pathology. The etiology of both disorders is unknown, and complex combinations of multiple susceptibility genes and environmental factors are thought to be involved. Both disorders are characterized by ubiquitous alpha-synuclein aggregates in distinct regions and cell types of the central nervous system. In PD, alpha-synuclein-positive aggregates appear to be largely neuronal while in MSA oligodendroglial inclusions prevail. In PD patients, the alpha-synuclein pathology is thought to evolve in a rather regular pattern, starting in the brainstem and olfactory bulb and extending gradually onto the substantia nigra and ultimately the cerebral cortex while the cerebellum is largely spared. MSA pathology has not been graded in a similar way yet; neuropathological analyses revealed neurodegeneration and gliosis primarily in the brainstem, midbrain and basal ganglia and the cerebellum, while the cortex is largely spared. To identify disease-specific transcriptional patterns in MSA, we chose CNS regions differentially affected in MSA and PD for comparative gene expression profiling: putamen, cerebellum and occipital cortex. Four genes were regulated in both MSA and PD putamen and twelve in MSA and PD cerebellum. Regulated transcripts were validated using real-time quantitative RT-PCR, and immunohistochemistry was performed for the most significantly downregulated transcripts in MSA and PD putamen, GPR86 and RGS14, associated with G protein signaling and transcriptional regulation.

Arginine/lysine-rich motifs typically function as targeting signals for the translocation of proteins to the nucleus. Here, we demonstrate that such a motif consisting of four basic amino acids in the polyglutamine protein ataxin-3 (Atx-3) serves as a recognition site for the interaction with the molecular chaperone VCP. Through this interaction, VCP modulates the fibrillogenesis of pathogenic forms of Atx-3 in a concentration-dependent manner, with low concentrations of VCP stimulating fibrillogenesis and excess concentrations suppressing it. No such effect was observed with a mutant Atx-3 variant, which does not contain a functional VCP interaction motif. Strikingly, a stretch of four basic amino acids in the ubiquitin chain assembly factor E4B was also discovered to be critical for VCP binding, indicating that arginine/lysine-rich motifs might be generally utilized by VCP for the targeting of proteins. In vivo studies with Drosophila models confirmed that VCP selectively modulates aggregation and neurotoxicity induced by pathogenic Atx-3. Together, these results define the VCP-Atx-3 association as a potential target for therapeutic intervention and suggest that it might influence the progression of spinocerebellar ataxia type 3.

Many neurodegenerative diseases are caused by intracellular, aggregate-prone proteins, including polyglutamine-expanded huntingtin in Huntington's disease (HD) and mutant tau in fronto-temporal dementia/tauopathy. Previously, we showed that rapamycin, an autophagy inducer, enhances mutant huntingtin fragment clearance and attenuated toxicity. Here we show much wider applications for this approach. Rapamycin enhances the autophagic clearance of different proteins with long polyglutamines and a polyalanine-expanded protein, and reduces their toxicity. Rapamycin also reduces toxicity in Drosophila expressing wild-type or mutant forms of tau and these effects can be accounted for by reductions in insoluble tau. Thus, our studies suggest that the scope for rapamycin as a potential therapeutic in aggregate diseases may be much broader than HD or even polyglutamine diseases.

Epidemiological evidence suggests that nonsteroidal anti-inflammatory drugs (NSAIDs) decrease the risk for Alzheimer's disease (AD). Certain NSAIDs can activate the peroxisome proliferator-activated receptor-gamma (PPARgamma), which is a nuclear transcriptional regulator. Here we show that PPARgamma depletion potentiates beta-secretase [beta-site amyloid precursor protein cleaving enzyme (BACE1)] mRNA levels by increasing BACE1 gene promoter activity. Conversely, overexpression of PPARgamma, as well as NSAIDs and PPARgamma activators, reduced BACE1 gene promoter activity. These results suggested that PPARgamma could be a repressor of BACE1. We then identified a PPARgamma responsive element (PPRE) in the BACE1 gene promoter. Mutagenesis of the PPRE abolished the binding of PPARgamma to the PPRE and increased BACE1 gene promoter activity. Furthermore, proinflammatory cytokines decreased PPARgamma gene transcription, and this effect was supressed by NSAIDs. We also demonstrate that in vivo treatment with PPARgamma agonists increased PPARgamma and reduced BACE1 mRNA and intracellular beta-amyloid levels. Interestingly, brain extracts from AD patients showed decreased PPARgamma expression and binding to PPRE in the BACE1 gene promoter. Our data strongly support a major role of PPARgamma in the modulation of amyloid-beta generation by inflammation and suggest that the protective mechanism of NSAIDs in AD involves activation of PPARgamma and decreased BACE1 gene transcription.

Huntington's disease can be used as a model to study neurodegenerative disorders caused by aggregation-prone proteins. It has been proposed that the entrapment of transcription factors in aggregates plays an important role in pathogenesis. We now report that the transcriptional activity of CBP is already repressed in the early time points by soluble mutant huntingtin, whereas the histone acetylase activity of CBP/p300 is gradually diminished over time. Mutant huntingtin bound much stronger to CBP than normal huntingtin, possibly contributing to repression. Especially at the later time points, CBP protein level was gradually reduced via the proteasome pathway. In sharp contrast, p300 was unaffected by mutant huntingtin. This selective degradation of CBP was absent in spinocerebellar ataxia 3. Thus, mutant huntingtin specifically affects CBP and not p300 both at the early and later time points, via multiple mechanisms. In addition to the reduction of CBP, also the altered ratio of these closely related histone acetyl transferases may affect chromatin structure and transcription and thus contribute to neurodegeneration.

Spinocerebellar ataxia type 3 (SCA3) is a late-onset neurodegenerative disorder caused by the expansion of a polyglutamine tract within the gene product, ataxin-3. We have previously shown that mutant ataxin-3 causes upregulation of inflammatory genes in transgenic SCA3 cell lines and human SCA3 pontine neurons. We report here a complex pattern of transcriptional changes by microarray gene expression profiling and Northern blot analysis in a SCA3 cell model. Twenty-three differentially expressed genes involved in inflammatory reactions, nuclear transcription, and cell surface-associated processes were identified. The identified corresponding proteins were analyzed by immunohistochemistry in human disease and control brain tissue to evaluate their implication in SCA3 pathogenesis. In addition to several inflammatory mediators upregulated in mutant ataxin-3 expressing cell lines and pontine neurons of SCA3 patients, we identified a profound repression of genes encoding cell surface-associated proteins in cells overexpressing normal ataxin-3. Correspondingly, these genes were upregulated in mutant ataxin-3 expressing cell lines and in pontine neurons of SCA3 patients. These findings identify for the first time target genes transcriptionally regulated by normal ataxin-3 and support the hypothesis that both loss of normal ataxin-3 and gain of function through protein-protein interacting properties of mutant ataxin-3 contribute to SCA3 pathogenesis.

Machado-Joseph disease (MJD) is a progressive neurodegenerative disorder caused by expansion of a CAG motif within the translated region of the human MJD (hMJD) gene which has been mapped to chromosome 14q. In this study, the hMJD gene was identified in two overlapping bacterial artificial chromosome (BAC) clones and contained 11 exons resulting in a 6.14 kb transcript. The 5'-flanking region of the hMJD gene included a TATA-less promoter with GC-rich regions, a CCAAT box and multiple potential SP1 binding sites. Luciferase reporter assays performed in neuronal and non-neuronal human cell lines demonstrated a core promoter within the 200 bp region immediately upstream of the putative transcriptional start site (-89 according to the start codon). DNA-protein interactions defined by electrophoretic mobility shift assays (EMSA) revealed specific binding of nuclear proteins to the putative core promoter region.

The human oncogene bcl-2 exerts protective functions in numerous models of apoptotic cell death and increased oxidative stress. We investigated the effects of inducible bcl-2 overexpression on cellular survival and redox status in dopaminergic rat pheochromocytoma PC 12 cells. Induction of high-level expression of bcl-2 in PC 12 cells resulted in generation of oxidative stress and cessation of growth by cell cycle arrest. Cell cycle arrest in bcl-2-overexpressing PC 12 cells was prevented by an inhibitor of extracellular signal-related kinase (ERK 1/2) activation. Protective effects of bcl-2 expression against L-DOPA neurotoxicity decreased with increasing amounts of bcl-2. Furthermore, high-level bcl-2 overexpression sensitized cells towards oxidative stress and glutathione depletion. Our data suggest that bcl-2 expression is beneficial only in a limited gene dosage range and that high-level expression of bcl-2 exerts potential deleterious effects.

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine disorder caused by a CAG repeat expansion in the coding region of a gene encoding ataxin-3, a protein of yet unknown function. Based on a comprehensive computational analysis, we propose a structural model and structure-based functions for ataxin-3. Our predictive strategy comprises the compilation of multiple sequence and structure alignments of carefully selected proteins related to ataxin-3. These alignments are consistent with additional information on sequence motifs, secondary structure, and domain architectures. The application of complementary methods revealed the homology of ataxin-3 to ENTH and VHS domain proteins involved in membrane trafficking and regulatory adaptor functions. We modeled the structure of ataxin-3 using the adaptin AP180 as a template and assessed the reliability of the model by comparison with known sequence and structural features. We could further infer potential functions of ataxin-3 in agreement with known experimental data. Our database searches also identified an as yet uncharacterized family of proteins, which we named josephins because of their pronounced homology to the Josephin domain of ataxin-3., (Copyright 2002 Wiley-Liss, Inc.)

Members of the Bcl-2 family of proteins function either to promote or to repress apoptosis. Bcl-2 has been mainly localised to the mitochondria and acts predominantly upstream of cytochrome c release in its prevention of apoptosis. Little is known about the function of Bcl-2 independent of an apoptotic stimulus. Here we demonstrate that inducible overexpression of the anti-apoptotic protein Bcl-2 in a PC12 Tet-on- cell line up-regulates mRNA expression and leads to phosphorylation of c-Jun at Ser73 via the ERK pathway in a time and concentration dependent manner. Phosphorylation of c-Jun was inhibited by the addition of the selective ERK inhibitor PD 98059. No activation of the stress-activated protein kinases JNK and p38 could be detected. This is the first evidence of a direct activation of the Ras-Raf-MAPK cascade by an anti-apoptotic protein. We propose that the selective activation of Ras, the ERK pathway and the subsequent phosphorylation of c-Jun contribute to the anti-apoptotic action of Bcl-2.

Variable expression and function of the cytochrome P4502D6 (CYP2D6) leads to distinct phenotypes termed ultrarapid (UM), extensive (EM), intermediate (IM) and poor metabolizer (PM). Whereas the PM phenotype is known to be caused by two null-alleles leading to absence of functional CYP2D6 protein, the large variability among individuals with functional alleles remained largely unexplained. In this study, we systematically investigated 76 liver biopsies from individuals with known sparteine metabolic ratios (MRS) for the relationships between CYP2D6 genotype, microsomal protein expression, bufuralol 1'-hydroxylase activity and in-vivo phenotype. Average CYP2D6 protein levels ranged from undetectable in PMs (MRS > 20) to 2.6 +/- 2.7 pmol/mg microsomal protein in IMs (1.2 < MRS< 20), 7.6 +/- 4.7 in EMs (0.2 < MRS < 1.2) and 23.8 +/- 7.7 in UMs (MRS < 0.2), respectively. Analysis with respect to genotype demonstrated gradually increased expression and function for individuals with no, one, two or three functional gene copies per genome. The recently discovered -1584 C/G promoter polymorphism was identified as another major factor for expression and function with the mutant [-1584G] promoter type being consistently associated with significantly higher expression than [-1584C]. To investigate functional differences between the detected variant protein forms CYP2D6.1, 2D6.2, 2D6.9 and 2D6.10, we expressed them recombinantly in insect cells. The most significant difference was a decrease in the relative P450 holoprotein content of all allelic forms, including the common functional variant 2D6.2, in comparison to 2D6.1, whereas only modest Km changes were observed. Taken together, these data provide further insight into the complex mechanisms that govern the highly variable expression and function of CYP2D6.

Spinocerebellar ataxia type 3 (SCA3) is a polyglutamine disorder caused by a CAG repeat expansion in the coding region of a gene encoding ataxin-3. To study putative alterations of gene expression induced by expanded ataxin-3, we performed PCR-based cDNA subtractive hybridization in a cell culture model of SCA3. In rat mesencephalic CSM14.1 cells stably expressing expanded ataxin-3, we found a significant upregulation of mRNAs encoding the endopeptidase matrix metalloproteinase 2 (MMP-2), the transmembrane protein amyloid precursor protein, the interleukin-1 receptor-related Fos-inducible transcript, and the cytokine stromal cell-derived factor 1alpha (SDF1alpha). Immunohistochemical studies of the corresponding or associated proteins in human SCA3 brain tissue confirmed these findings, showing increased expression of MMP-2 and amyloid beta-protein (Abeta) in pontine neurons containing nuclear inclusions. In addition, extracellular Abeta-immunoreactive deposits were detected in human SCA3 pons. Furthermore, pontine neurons of SCA3 brains strongly expressed the antiinflammatory interleukin-1 receptor antagonist, the proinflammatory cytokine interleukin-1beta, and the proinflammatory chemokine SDF1. Finally, increased numbers of reactive astrocytes and activated microglial cells were found in SCA3 pons. These results suggest that inflammatory processes are involved in the pathogenesis of SCA3.

Bcl-2 has been shown to exert its antiapoptotic activity predominantly at the level of mitochondria by preventing cytochrome c release. Whether Bcl-2 is involved in the regulation of mitochondrial function prior to an apoptotic stimulus remains elusive. Using functional and spectrophotometric measurements in an inducible PC12-Tet-on-bcl-2 cell line we demonstrate that induction of Bcl-2 overexpression rapidly reduced cytochrome b and c levels as well as complex I activity. To confirm that these changes were specific for Bcl-2 we generated a bcl-2 antisense construct under the control of the tetracycline responsive promotor. Transient transfection with this antisense plasmid prevented both the decrease of cytochrome b and c levels and the loss of complex I activity. The decrease of cytochrome b levels was paralleled by a decrease of cytochrome b mRNA levels while Northern blot analysis of cytochrome c mRNA expression did not reveal any overt changes in Bcl-2 cells. We propose that the antiapoptotic properties of Bcl-2 are related to the reduction of mitochondrial complex I activity and lowered mitochondrial cytochrome b and c levels., (Copyright 2001 Academic Press.)