Safety and potential efficacy of gemfibrozil as a supportive treatment for children with late infantile neuronal ceroid lipofuscinosis and other lipid storage disorders.

Neuronal Ceroid Lipofuscinosis (NCL), also known as Batten disease, is a group of genetically distinct lysosomal disorders that mainly affect the central nervous system, resulting in progressive motor and cognitive decline primarily in children. Multiple distinct genes involved in the metabolism of lipids have been identified to date with various mutations in this family of diseases. There is no cure for these diseases but some new therapeutic approaches have been tested that offer more hope than the standard palliative care. Many of the therapeutic advances require invasive procedures but some progress in slowing the disease has been found and more options can be expected in the future. We also review the literature on children with disease/conditions other than NCL for the non-invasive use, safety, and tolerability of a lipid-lowering drug, gemfibrozil, as a potential treatment for NCLs. Gemfibrozil has shown efficacy in an animal model of NCL known as CLN2 (late infantile classic juvenile) and has been shown to be safe for lowering lipids in children. Among the 200 non-NCL children found in the published literature who were treated with gemfibrozil for NCL-related problems, only 3 experienced adverse events, including 2 with muscle pain and 1 with localized linear IgA bullous dermatitis. We conclude that gemfibrozil is safe for long-term use in children, causes minimal adverse events, is well tolerated, and may delay the progression of NCLs. Gemfibrozil may potentially be an alternative to more invasive therapeutic approaches currently under investigation and has the potential to be used in combination with other therapeutic approaches.

Mechanical loading differentially regulates membrane-bound and soluble RANKL availability in MC3T3-E1 cells.

To understand the biochemical response of RANKL in response to mechanical loading, MC3T3-E1 cells were biequiaxially stretched. A murine RANKL cDNA with double epitopes, pEF6 HA-RANKL-V5His, was transfected into MC3T3-E1 cells, which were then stretched. Endogenous RANKL protein expression increased in response to mechanical loading. Membrane-bound RANKL (HA-RANKL-V5His) increased in cell lysates while soluble RANKL (RANKL-V5His) decreased in the conditioned media after mechanical loading. This may have resulted from the decreased activity of TACE after mechanical loading. Increased membrane-bound RANKL may be one of the mechanisms through which osteoblasts adapt to mechanical loading by regulating osteoclastogenic activity in a region-specific manner.

Stromal cell-derived factor 1 (SDF-1, CXCL12) is increased in subacromial bursitis and downregulated by steroid and nonsteroidal anti-inflammatory agents.

Several studies have demonstrated that inflammation in the subacromial bursa is an important component in the pathogenesis of impingement syndrome. We have demonstrated in a previous study that many inflammatory cytokines, including stromal cell-derived factor 1 (SDF-1, CXCL12), are increased in the subacromial bursa [Blaine et al. 2005. J Shoulder Elbow Surg 14(Suppl 1):84S-89S]. SDF-1 is a potent chemotactic and angiogenic factor that stimulates recruitment of inflammatory cells. In the current study, we proposed that the resident cells in subacromial bursal tissue produce SDF-1, which can play a role in the inflammatory reponse of bursal tissue, and that this chemokine can be regulated by steroid (dexamethasone) and nonsteroidal anti-inflammatory medications (NSAIDs). Twenty-two subacromial bursa tissues (18 bursitis and 4 normal bursa) were obtained intraoperatively from patients during shoulder surgery and analyzed using the cDNA Array technique in accordance with an IRB approved protocol. cDNA array results were confirmed with real-time reverse transcription-polymerase chain reaction (RT-PCR). Bursal cells (from 4 normal bursa, 3 bursitis) and two normal bone marrow with whole tissue explants were cultured for one passage. Cell culture supernatants were collected and SDF-1 protein was detected with enzyme-linked immunosorbent assay (ELISA). Cultured bursal cells were treated with a COX-2 inhibitor and dexamethasone, and cells was harvested at 1-day and 4-day intervals. SDF-1 expression was evaluated by real-time RT-PCR and ELISA. cDNA Array analysis demonstrated that the gene expression of SDF-1 was increased in patients with subacromial bursitis compared to controls (p < 0.05). Real-time RT-PCR also revealed that the mRNA expression of SDF-1 in bursitis tissue is increased 10-fold over control tissue. While the normal bursal cells produced negligible amounts of SDF-1 protein, cultured cells derived from bursitis lesion released as much SDF-1 protein (235 pg/100,000 cells) as normal bone marrow stromal cells (283 pg/100,000 cells) as measured by ELISA. The addition of a COX-2 inhibitor and dexamethasone to bursitis cell lines led to decreased SDF-1 expression levels compared to untreated bursitis cell lines. These studies demonstrate that there is a significant elevation of SDF-1 expression in the subacromial bursa of patients with rotator cuff disease. Furthermore, this chemokine can be downregulated by COX-2 inhibitors and steroids. These results provide biologic evidence for the use of steroid and NSAIDs in the treatment of subacromial bursitis. In the future, targeted inhibition of molecules such as SDF-1 in the subacromial bursa may present a therapeutic strategy that may avoid the side effects of these other (steroid and NSAID) medications.

Zoledronate induces apoptosis in cells from fibro-cellular membrane of unicameral bone cyst (UBC).

Unicameral bone cyst (UBC) is a benign cystic lesion in children which is prone to fracture. Various treatments are available, but recurrence after different types of percutaneous injection therapy can cause bone destruction and pathologic fracture. The potential therapeutic effects of anti-resorptive agents, such as bisphosphonates, have not been investigated for UBC. The objective of this study was to characterize the cells from the fibro-cellular membrane of unicameral bone cyst (UBC cells) and to determine whether zoledronate, a nitrogen-containing bisphosphonate, could induce apoptosis in UBC cells. Flow cytometry and immunoblotting were performed in order to determine whether zoledronate induced apoptosis. Cells derived from normal human trabecular bones were used as controls against UBC cells to compare the effect of zoledronate in inducing apoptosis. Immunohisto/cytochemistry (IHC/ICC) and mini-array analyses were performed on tissues and cultured cells. Isolated peripheral blood mononuclear cells were incubated with conditioned media from the UBC cells to determine whether they are capable of inducing osteoclastogenesis. UBC membrane is composed of cells staining positively with CD68, SDF-1, STRO-1 and RANKL, but in vitro cells showed no staining with antibodies to CD68 and STRO-1, suggesting that there was a clonal selection of stromal cells during cell culture. UBC cells also express RUNX2 (runt-related transcription factor-2, core binding factor-1), a key transcription factor for osteoblastic differentiation. In addition, media collected from UBC cells induced a generation of multi-nucleated osteoclast-like cells of peripheral blood mononuclear cells. Zoledronate induced apoptosis of UBC cells in a dose-dependent manner. Apoptosis was evidenced by induction of the active cleaved form of caspase-3. The baseline apoptotic fractions were similar in UBC cells and trabecular bone cells. However, in the overall apoptotic fractions in this study, trabecular bone cells showed 17.2% of apoptosis, significantly lower than 24.2% of UBC cells (p-value=0.007). With the various zoledronate concentrations, mean apoptotic fractions of trabecular bone cells was 19.2%, significantly lower than 27.8% of UBC cells (p-value=0.040). With GGOH co-treatment in various zoledronate concentrations, 15.1% apoptosis was shown in trabecular bone cells, which was not significantly lower than 20.6% of UBC cells (p-value=0.076). This data suggests that zoledronate causes apoptosis in both UBC and trabecular bone cells by inhibition of the mevalonate pathway. In addition to the known anti-osteoclastogenic effect of bisphosphonates, the GGOH inhibitory effects of zoledronate were more prominent in UBC cells than trabecular bone cells, indicating their potential therapeutic role in UBC.

mu-Calpain regulates receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis via NF-kappaB activation in RAW 264.7 cells.

To clarify the role of calpain in the receptor activator of NF-kappaB ligand (RANKL)-supported osteoclastogenesis, RANKL-induced calpain activation was examined by using murine RAW 264.7 cells and bone marrow-derived monocyte/macrophage progenitors. We found that calpain activity increased in response to RANKL in both cell types based on alpha-spectrinolysis and that mu-calpain, rather than m-calpain, was activated during RANKL-supported osteoclastogenesis in RAW 264.7 cells. Overexpression of mu-calpain clearly augmented RANKL-supported osteoclastogenesis in RAW 264.7 cells, thereby implicating its pivotal role in this process. Cell-permeable calpain inhibitors, including calpastatin and calpeptin, were sufficient to suppress RANKL-supported osteoclastogenesis based on decreased expression of the osteoclastogenic marker, matrix metalloproteinase 9, and the generation of tartrate-resistant acid phosphatase-positive multinucleated cells in both cell types. Calpain inhibitors suppressed NF-kappaB activation via inhibition of the cleavage of inhibitor of NF-kappaB(IkappaBalpha)in RAW 264.7 cells. Taken together, our findings suggest that mu-calpain is essential to the regulation of RANKL-supported osteoclastogenesis via NF-kappaB activation.

PEN-2 and APH-1 coordinately regulate proteolytic processing of presenilin 1.

Presenilin (PS, PS1/PS2) complexes are known to be responsible for the intramembranous gamma-secretase cleavage of the beta-amyloid precursor protein and signaling receptor Notch. PS holoprotein undergoes endoproteolysis by an unknown enzymatic activity to generate NH(2)- and COOH-terminal fragments, a process that is required for the formation of the active and stable PS/-gamma-secretase complex. Biochemical and genetic studies have recently identified nicastrin, APH-1, and PEN-2 as essential cofactors that physically interact with PS1 and are necessary for the gamma-secretase activity. However, their precise function in regulating the PS complex and gamma-secretase activity remains unknown. Here, we demonstrate that endogenous PEN-2 preferentially interacts with PS1 holoprotein. Down-regulation of PEN-2 expression by small interfering RNA (siRNA) abolishes the endoproteolysis of PS1, whereas overexpression of PEN-2 promotes the production of PS1 fragments, indicating a critical role for PEN-2 in PS1 endoproteolysis. Interestingly, accumulation of full-length PS1 resulting from down-regulation of PEN-2 is alleviated by additional siRNA down-regulation of APH-1. Furthermore, overexpression of APH-1 facilitates PEN-2-mediated PS1 proteolysis, resulting in a significant increase in PS1 fragments. Our data reveal a direct role of PEN-2 in proteolytic cleavage of PS1 and a regulatory function of APH-1, in coordination with PEN-2, in the biogenesis of the PS1 complex.

In vivo role of caspases in excitotoxic neuronal death: generation and analysis of transgenic mice expressing baculoviral caspase inhibitor, p35, in postnatal neurons.

Caspases, a family of cysteine proteases, are thought to be critical mediators of apoptosis. To examine the role of neuronal caspases in excitotoxic neurodegeneration in vivo, we have generated transgenic mice expressing the baculovirus protein p35, a potent viral caspase inhibitor, using the neuron-specific calmodulin dependent kinase-II alpha (CaMKII-alpha) promoter. The expression of p35 was confirmed by reverse transcriptase-polymerase chain reaction (RT-PCR), Western blotting and immunohistochemistry. We analyzed caspase activation and cell death by employing an experimental paradigm, in which the excitotoxin kainate (KA) was injected into CA1 of hippocampus and the distribution of the caspase-generated actin fragment was detected immunohistochemically. While kainate treatment led to selective neuronal death in the CA1, CA3 and CA4 of non-transgenic control mice, we observed restricted caspase activation only in the CA3 sector. The transgenic expression of p35 consistently inhibited the kainate-induced caspase activation, but failed to influence the death of neurons to any extent. In addition, we observed concomitant early calpain activation in the specific areas where neurons underwent degeneration in both the transgenic and non-transgenic mice. These results indicate that p35-inhibitable caspases play rather minor roles in the kainate-induced excitotoxicity and that the relative contribution of calpain is likely to be greater than that of caspases.

Generation of amyloid beta peptide with pyroglutamate at position 3 in primary cortical neurons.

The majority of amyloid beta peptide (Abeta) deposited in the brains of Alzheimer's disease (AD) patients is N-truncated, especially Abeta starting with pyroglutamate at position 3 (Abeta(3(pE))). To develop a system in which Abeta(3(pE)) is generated in primary neurons and to clarify the cyclization mechanism of N-terminal glutamate, we constructed amyloid precursor protein complementary DNAs which encoded a potential precursor for Abeta(3(pE)) by amino acid substitution and deletion. Among them, expression of NLQ construct by Sindbis virus resulted in secretion of Abeta(3(pE)) from primary neurons, whereas the N-termini of Abeta derived from NL and NLE constructs were intact. Therefore, the NLQ construct would be useful in establishing an animal model which produces Abeta(3(pE)).

Presenilin-dependent gamma-secretase-like intramembrane cleavage of ErbB4.

An unusual protease gamma-secretase requires functional presenilins and cleaves substrates (e.g. amyloid beta-protein precursor and Notch) with very loose amino acid sequence specificity within the transmembrane region. Here we report that ErbB4, a tyrosine kinase receptor for neuregulins, is a substrate for presenilin-dependent gamma-secretase. Our studies show that constitutive ectodomain shedding of full-length ErbB4 yields the approximately 80-kDa membrane-associated C-terminal fragment (B4-CTF). Subsequent intramembrane cleavage of the B4-CTF was inhibited in the cells devoid of functional presenilins or by treatment of cells with a gamma-secretase inhibitor, leading to enhanced accumulation of B4-CTF. Furthermore, an in vitro gamma-secretase assay demonstrated that the intracellular domain of ErbB4 (B4-ICD) was produced and subsequently released into the soluble fraction in a presenilin-dependent manner. We have also shown that ectopically expressed B4-ICD is localized to the nucleus, suggesting that the presenilin-dependent cleavage of ErbB4 generates the soluble B4-ICD that functions in the nucleus presumably at transcriptional level. Our study indicates that ErbB4 represents a first receptor tyrosine kinase that undergoes intramembrane proteolysis and may mediate a novel signaling function independent of its canonical role as a receptor tyrosine kinase. Our studies also support the idea that presenilins play a generic role in intramembrane cleavage of selected type I membrane proteins.