Frontotemporal lobar degeneration (FTLD) is a devastating neurodegenerative disease that is the second most common form of dementia affecting individuals under age 65. The most common pathological subtype, FTLD with transactive response DNA-binding protein with a molecular weight of 43 kDa inclusions (FTLD-TDP), is often caused by autosomal dominant mutations in the progranulin gene (GRN) encoding the progranulin protein (PGRN). GRN pathogenic mutations result in haploinsufficiency, usually by nonsense-mediated decay of the mRNA. Since the discovery of these mutations in 2006, several groups have published data and animal models that provide further insight into the genetic and functional relevance of PGRN in the context of FTLD-TDP. These studies were critical in initiating our understanding of the role of PGRN in neural development, degeneration, synaptic transmission, cell signaling, and behavior. Furthermore, recent publications have now identified the receptors for PGRN, which will hopefully lead to additional therapeutic targets. Additionally, drug screens have been conducted to identify pharmacological regulators of PGRN levels to be used as potential treatments for PGRN haploinsufficiency. Here we review recent literature describing relevant data on GRN genetics, cell culture experiments describing the potential role and regulators of PGRN in the central nervous system, animal models of PGRN deficiency, and potential PGRN-related FTLD therapies that are currently underway. The present review aims to underscore the necessity of further elucidation of PGRN biology in FTLD-related neurodegeneration.

Background

Progranulin (PGRN), a widely secreted growth factor, is involved in multiple biological functions, and mutations located within the PGRN gene (GRN) are a major cause of frontotemporal lobar degeneration with TDP-43-positive inclusions (FLTD-TDP). In light of recent reports suggesting PGRN functions as a protective neurotrophic factor and that sortilin (SORT1) is a neuronal receptor for PGRN, we used a Sort1-deficient (Sort1−/−) murine primary hippocampal neuron model to investigate whether PGRN’s neurotrophic effects are dependent on SORT1. We sought to elucidate this relationship to determine what role SORT1, as a regulator of PGRN levels, plays in modulating PGRN’s neurotrophic effects.

Results

As the first group to evaluate the effect of PGRN loss in Grn knockout primary neuronal cultures, we show neurite outgrowth and branching are significantly decreased in Grn−/− neurons compared to wild-type (WT) neurons. More importantly, we also demonstrate that PGRN overexpression can rescue this phenotype. However, the recovery in outgrowth is not observed following treatment with recombinant PGRN harboring missense mutations p.C139R, p.P248L or p.R432C, indicating that these mutations adversely affect the neurotrophic properties of PGRN. In addition, we also present evidence that cleavage of full-length PGRN into granulin peptides is required for increased neuronal outgrowth, suggesting that the neurotrophic functions of PGRN are contained within certain granulins. To further characterize the mechanism by which PGRN impacts neuronal morphology, we assessed the involvement of SORT1. We demonstrate that PGRN induced-outgrowth occurs in the absence of SORT1 in Sort1−/− cultures.

Conclusion

We demonstrate that loss of PGRN impairs proper neurite outgrowth and branching, and that exogenous PGRN alleviates this impairment. Furthermore, we determined that exogenous PGRN induces outgrowth independent of SORT1, suggesting another receptor(s) is involved in PGRN induced neuronal outgrowth.

Cisplatin is a platinum-based chemotherapeutic agent that induces peripheral neuropathy in 30% of patients. Peripheral neuropathy is the dose limiting side effect, which has no preventative therapy. We have previously shown that cisplatin induces apoptosis in dorsal root ganglion (DRG) sensory neurons by covalently binding to nuclear DNA (nDNA), resulting in DNA damage, subsequent p53 activation and Bax-mediated apoptosis via the mitochondria. We now demonstrate that cisplatin also directly binds to mitochondrial DNA (mtDNA) with the same binding affinity as nDNA. Cisplatin binds 1 platinum molecule per 2166 mtDNA base pairs and 1 platinum molecule per 3800 nDNA base pairs. Furthermore, cisplatin treatment inhibits mtDNA replication as detected by 5-bromo-2'-deoxy-uridine (BrdU) incorporation and inhibits transcription of mitochondrial genes. The relative reduction in mtDNA transcription is directly related to the distance the gene is located from the transcription initiation point, which implies that randomly formed platinum adducts block transcription. Cisplatin treated DRG neurons exhibit mitochondrial vacuolization and degradation in vitro and in vivo. Taken together, this data suggests that direct mtDNA damage may provide a novel, distinct mechanism for cisplatin-induced neurotoxicity separate from the established nDNA damage pathway.

Background. Sentinel node biopsy (SNB) represents the standard of care in breast cancer axillary evaluation. Our study aims to characterize the patterns of malignant cell distribution within the sentinel nodes (SN). Methods. In a retrospective IRB-approved study, we examined the anatomic location of the nodal area with the highest radioactive signal or most intense blue staining (hot spot) and its distance from the metastatic foci. Results. 58 patients underwent SNB between January 2006 and February 2007. 12 patients with 19 positive SN were suitable for analysis. 4 (21%) metastases were located in the nodal hilum and 15 (79%) in the cortex. 6 (31%) metastases were found adjacent to the hotspot, and 9 (47%) within 4 mm of the hotspot. Conclusions. In our pilot series, SN metastases were within 4 mm of the hotspot in 78% of the cases. Pathologic analysis focused in that area may contribute to the more accurate identification of nodal metastases.

Background

Covalent linkage of ubiquitin regulates the function and, ultimately, the degradation of many proteins by the ubiquitin-proteasome system (UPS). Given its essential role in protein regulation, even slight perturbations in UPS activity can substantially impair cellular function.

Methodology/Principal Findings

We have generated and characterized a novel transgenic mouse model which expresses a previously described reporter for UPS function. This UPS reporter contains a degron sequence attached to the C-terminus of green fluorescent protein, and is predominantly expressed in neurons throughout the brain of our transgenic model. We then demonstrated that this reporter system is sensitive to UPS inhibition in vivo.

Conclusions/Significance

Given the obstacles associated with evaluating proteasomal function in the brain, our mouse model uniquely provides the capability to monitor UPS function in real time in individual neurons of a complex organism. Our novel mouse model now provides a useful resource with which to evaluate the impact of aging, as well as various genetic and/or pharmacological modifiers of neurodegenerative disease(s).

When B-lymphocytes differentiate into plasma cells, immunoglobulin (Ig) heavy and light chain synthesis escalates and the entire secretory apparatus expands to support high-rate antibody secretion. These same events occur when murine B-cells are stimulated with lipopolysaccharide (LPS), providing an in vitro model in which to investigate the differentiation process. The unfolded protein response (UPR), a multi-pathway signaling response emanating from the endoplasmic reticulum (ER) membrane, allows cells to adapt to increasing demands on the protein folding capacity of the ER. As such, the UPR plays a pivotal role in the differentiation of antibody-secreting cells. Three specific stress sensors, IRE1, PERK/PEK and ATF6, are central to the recognition of ER stress and induction of the UPR. IRE1 triggers splicing of Xbp-1 mRNA, yielding a transcriptional activator of the UPR termed XBP-1(S), and activation of the IRE1/XBP-1 pathway has been reported to be required for expansion of the ER and antibody secretion. Here, we provide evidence that PERK is not activated in LPS-stimulated splenic B-cells, whereas XBP-1(S) and the UPR transcriptional activator ATF6 are both induced. We further demonstrate that Perk-/- B-cells develop and are fully competent for induction of Ig synthesis and antibody secretion when stimulated with LPS. These data provide clear evidence for differential activation and utilization of distinct UPR components as activated B-lymphocytes increase Ig synthesis and differentiate into specialized secretory cells.

The TAR DNA-binding protein 43 (TDP-43) has been identified as the major disease protein in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U), defining a novel class of neurodegenerative conditions: the TDP-43 proteinopathies. The first pathogenic mutations in the gene encoding TDP-43 (TARDBP) were recently reported in familial and sporadic ALS patients, supporting a direct role for TDP-43 in neurodegeneration. In this study, we report the identification and functional analyses of two novel and one known mutation in TARDBP that we identified as a result of extensive mutation analyses in a cohort of 296 patients with variable neurodegenerative diseases associated with TDP-43 histopathology. Three different heterozygous missense mutations in exon 6 of TARDBP (p.M337V, p.N345K, and p.I383V) were identified in the analysis of 92 familial ALS patients (3.3%), while no mutations were detected in 24 patients with sporadic ALS or 180 patients with other TDP-43–positive neurodegenerative diseases. The presence of p.M337V, p.N345K, and p.I383V was excluded in 825 controls and 652 additional sporadic ALS patients. All three mutations affect highly conserved amino acid residues in the C-terminal part of TDP-43 known to be involved in protein-protein interactions. Biochemical analysis of TDP-43 in ALS patient cell lines revealed a substantial increase in caspase cleaved fragments, including the ∼25 kDa fragment, compared to control cell lines. Our findings support TARDBP mutations as a cause of ALS. Based on the specific C-terminal location of the mutations and the accumulation of a smaller C-terminal fragment, we speculate that TARDBP mutations may cause a toxic gain of function through novel protein interactions or intracellular accumulation of TDP-43 fragments leading to apoptosis.

Author Summary

The abnormal accumulation of disease proteins in neuronal cells of the brain is a characteristic feature of many neurodegenerative diseases. Rare mutations in the genes that encode the accumulating proteins have been identified in these disorders and are crucial for the development of cell and animal models used to study neurodegeneration. Recently, the TAR DNA-binding protein 43 (TDP-43) was identified as the disease accumulating protein in patients with frontotemporal lobar degeneration with ubiquitin inclusions (FTLD-U) and in amyotrophic lateral sclerosis (ALS). TDP-43 was also found in the brains of 20–30% of patients with Alzheimer's disease (AD). Here, we evaluated whether mutations in TDP-43 cause disease in a cohort of 296 patients presenting with FTLD, ALS or AD. We identified three missense mutations in three out of 92 familial ALS patients (3.3%), and no mutations in AD or FTLD patients. All the identified mutations clustered in exon 6, which codes for a highly conserved region in the C-terminal part of the TDP-43 protein, which is known to be involved in the interaction of TDP-43 with other proteins. We conclude that mutations in TDP-43 are a rare cause of familial ALS, but so far are not found in other neurodegenerative diseases.