Making the connection - shared molecular machinery and evolutionary links underlie the formation and plasticity of occluding junctions and synapses.

The pleated septate junction (pSJ), an ancient structure for cell-cell contact in invertebrate epithelia, has protein components that are found in three more-recent junctional structures, the neuronal synapse, the paranodal region of the myelinated axon and the vertebrate epithelial tight junction. These more-recent structures appear to have evolved through alterations of the ancestral septate junction. During its formation in the developing animal, the pSJ exhibits plasticity, although the final structure is extremely robust. Similar to the immature pSJ, the synapse and tight junctions both exhibit plasticity, and we consider evidence that this plasticity comes at least in part from the interaction of members of the immunoglobulin cell adhesion molecule superfamily with highly regulated membrane-associated guanylate kinases. This plasticity regulation probably arose in order to modulate the ancestral pSJ and is maintained in the derived structures; we suggest that it would be beneficial when studying plasticity of one of these structures to consider the literature on the others. Finally, looking beyond the junctions, we highlight parallels between epithelial and synaptic membranes, which both show a polarized distribution of many of the same proteins - evidence that determinants of apicobasal polarity in epithelia also participate in patterning of the synapse.

Activity-dependent secretion of progranulin from synapses.

The secreted growth factor progranulin (PGRN) has been shown to be important for regulating neuronal survival and outgrowth, as well as synapse formation and function. Mutations in the PGRN gene that result in PGRN haploinsufficiency have been identified as a major cause of frontotemporal dementia (FTD). Here we demonstrate that PGRN is colocalized with dense-core vesicle markers and is co-transported with brain-derived neurotrophic factor (BDNF) within axons and dendrites of cultured hippocampal neurons in both anterograde and retrograde directions. We also show that PGRN is secreted in an activity-dependent manner from synaptic and extrasynaptic sites, and that the temporal profiles of secretion are distinct in axons and dendrites. Neuronal activity is also shown to increase the recruitment of PGRN to synapses and to enhance the density of PGRN clusters along axons. Finally, treatment of neurons with recombinant PGRN is shown to increase synapse density, while decreasing the size of the presynaptic compartment and specifically the number of synaptic vesicles per synapse. Together, this indicates that activity-dependent secretion of PGRN can regulate synapse number and structure.

Stress-mediated aggregation of disease-associated proteins in amyloid bodies.

The formation of protein aggregates is a hallmark of many neurodegenerative diseases and systemic amyloidoses. These disorders are associated with the fibrillation of a variety of proteins/peptides, which ultimately leads to cell toxicity and tissue damage. Understanding how amyloid aggregation occurs and developing compounds that impair this process is a major challenge in the health science community. Here, we demonstrate that pathogenic proteins associated with Alzheimer's disease, diabetes, AL/AA amyloidosis, and amyotrophic lateral sclerosis can aggregate within stress-inducible physiological amyloid-based structures, termed amyloid bodies (A-bodies). Using a limited collection of small molecule inhibitors, we found that diclofenac could repress amyloid aggregation of the ß-amyloid (1-42) in a cellular setting, despite having no effect in the classic Thioflavin T (ThT) in vitro fibrillation assay. Mapping the mechanism of the diclofenac-mediated repression indicated that dysregulation of cyclooxygenases and the prostaglandin synthesis pathway was potentially responsible for this effect. Together, this work suggests that the A-body machinery may be linked to a subset of pathological amyloidosis, and highlights the utility of this model system in the identification of new small molecules that could treat these debilitating diseases.

Drosophila adducin regulates Dlg phosphorylation and targeting of Dlg to the synapse and epithelial membrane.

Adducin is a cytoskeletal protein having regulatory roles that involve actin filaments, functions that are inhibited by phosphorylation of adducin by protein kinase C. Adducin is hyperphosphorylated in nervous system tissue in patients with the neurodegenerative disease amyotrophic lateral sclerosis, and mice lacking ß-adducin have impaired synaptic plasticity and learning. We have found that Drosophila adducin, encoded by hu-li tai shao (hts), is localized to the post-synaptic larval neuromuscular junction (NMJ) in a complex with the scaffolding protein Discs large (Dlg), a regulator of synaptic plasticity during growth of the NMJ. hts mutant NMJs are underdeveloped, whereas over-expression of Hts promotes Dlg phosphorylation, delocalizes Dlg away from the NMJ, and causes NMJ overgrowth. Dlg is a component of septate junctions at the lateral membrane of epithelial cells, and we show that Hts regulates Dlg localization in the amnioserosa, an embryonic epithelium, and that embryos doubly mutant for hts and dlg exhibit defects in epithelial morphogenesis. The phosphorylation of Dlg by the kinases PAR-1 and CaMKII has been shown to disrupt Dlg targeting to the NMJ and we present evidence that Hts regulates Dlg targeting to the NMJ in muscle and the lateral membrane of epithelial cells by controlling the protein levels of PAR-1 and CaMKII, and consequently the extent of Dlg phosphorylation.

Clinical and pathological features of amyotrophic lateral sclerosis caused by mutation in the C9ORF72 gene on chromosome 9p.

Two studies recently identified a GGGGCC hexanucleotide repeat expansion in a non-coding region of the chromosome 9 open-reading frame 72 gene (C9ORF72) as the cause of chromosome 9p-linked amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). In a cohort of 231 probands with ALS, we identified the C9ORF72 mutation in 17 familial (27.4%) and six sporadic (3.6%) cases. Patients with the mutation presented with typical motor features of ALS, although subjects with the C9ORF72 mutation had more frequent bulbar onset, compared to those without this mutation. Dementia was significantly more common in ALS patients and families with the C9ORF72 mutation and was usually early-onset FTD. There was striking clinical heterogeneity among the members of individual families with the mutation. The associated neuropathology was a combination of ALS with TDP-ir inclusions and FTLD-TDP. In addition to TDP-43-immunoreactive pathology, a consistent and specific feature of cases with the C9ORF72 mutation was the presence of ubiquitin-positive, TDP-43-negative inclusions in a variety of neuroanatomical regions, such as the cerebellar cortex. These findings support the C9ORF72 mutation as an important newly recognized cause of ALS, provide a more detailed characterization of the associated clinical and pathological features and further demonstrate the clinical and molecular overlap between ALS and FTD.

Local self-renewal can sustain CNS microglia maintenance and function throughout adult life.

Microgliosis is a common response to multiple types of damage in the CNS. However, the origin of the cells involved in this process is still controversial and the relative importance of local expansion versus recruitment of microglia progenitors from the bloodstream is unclear. Here, we investigated the origin of microglia using chimeric animals obtained by parabiosis. We found no evidence of microglia progenitor recruitment from the circulation in denervation or CNS neurodegenerative disease, suggesting that maintenance and local expansion of microglia are solely dependent on the self-renewal of CNS resident cells in these models.

20-hydroxyecdysone (20E) signaling regulates amnioserosa morphogenesis during Drosophila dorsal closure: EcR modulates gene expression in a complex with the AP-1 subunit, Jun.

Steroid hormones influence diverse biological processes throughout the animal life cycle, including metabolism, stress resistance, reproduction, and lifespan. In insects, the steroid hormone, 20-hydroxyecdysone (20E), is the central hormone regulator of molting and metamorphosis, and plays roles in tissue morphogenesis. For example, amnioserosa contraction, which is a major driving force in Drosophila dorsal closure (DC), is defective in embryos mutant for 20E biosynthesis. Here, we show that 20E signaling modulates the transcription of several DC participants in the amnioserosa and other dorsal tissues during late embryonic development, including zipper, which encodes for non-muscle myosin. Canonical ecdysone signaling typically involves the binding of Ecdysone receptor (EcR) and Ultraspiracle heterodimers to ecdysone-response elements (EcREs) within the promoters of responsive genes to drive expression. During DC, however, we provide evidence that 20E signaling instead acts in parallel to the JNK cascade via a direct interaction between EcR and the AP-1 transcription factor subunit, Jun, which together binds to genomic regions containing AP-1 binding sites but no EcREs to control gene expression. Our work demonstrates a novel mode of action for 20E signaling in Drosophila that likely functions beyond DC, and may provide further insights into mammalian steroid hormone receptor interactions with AP-1.

Fus gene mutations in familial and sporadic amyotrophic lateral sclerosis.

Mutations in the fused in sarcoma (FUS) gene have recently been found to cause familial amyotrophic lateral sclerosis (FALS). We screened FUS in a cohort of 200 ALS patients [32 FALS and 168 sporadic ALS (SALS)]. In one FALS proband, we identified a mutation (p.R521C) that was also present in her affected daughter. Their clinical phenotype was remarkably similar and atypical of classic ALS, with symmetric proximal pelvic and pectoral weakness. Distal weakness and upper motor neuron features only developed late. Neuropathological examination demonstrated FUS-immunoreactive neuronal and glial inclusions in the spinal cord and many extramotor regions, but no TDP-43 pathology. We also identified a novel mutation (p.G187S) in one SALS patient. Overall, FUS mutations accounted for 3% of our non-SOD1, non-TARDBP FALS cases and 0.6% of SALS. This study demonstrates that the phenotype with FUS mutations extends beyond classical ALS cases. Our findings suggest there are specific clinicogenetic correlations and provide the first detailed neuropathological description.

Homeodomain-interacting protein kinase (Hipk) plays roles in nervous system and muscle structure and function.

Homeodomain-interacting protein kinases (Hipks) have been previously associated with cell proliferation and cancer, however, their effects in the nervous system are less well understood. We have used Drosophila melanogaster to evaluate the effects of altered Hipk expression on the nervous system and muscle. Using genetic manipulation of Hipk expression we demonstrate that knockdown and over-expression of Hipk produces early adult lethality, possibly due to the effects on the nervous system and muscle involvement. We find that optimal levels of Hipk are critical for the function of dopaminergic neurons and glial cells in the nervous system, as well as muscle. Furthermore, manipulation of Hipk affects the structure of the larval neuromuscular junction (NMJ) by promoting its growth. Hipk regulates the phosphorylation of the synapse-associated cytoskeletal protein Hu-li tai shao (Hts; adducin in mammals) and modulates the expression of two important protein kinases, Calcium-calmodulin protein kinase II (CaMKII) and Partitioning-defective 1 (PAR-1), all of which may alter neuromuscular structure/function and influence lethality. Hipk also modifies the levels of an important nuclear protein, TBPH, the fly orthologue of TAR DNA-binding protein 43 (TDP-43), which may have relevance for understanding motor neuron diseases.

Bone marrow-derived cells in the central nervous system of a mouse model of amyotrophic lateral sclerosis are associated with blood vessels and express CX(3)CR1.

Amyotrophic lateral sclerosis (ALS) is associated with increased numbers of microglia within the CNS. However, it is unclear to what extent bone marrow (BM)-derived cells contribute to this microgliosis. We have studied the adoptive transfer of green fluorescent protein (GFP)-labeled whole BM cells and BM from mice that express GFP only in CX(3)CR1+ cells (CX(3)CR1(+/GFP)) into the CNS of a murine model of ALS having over-expression of mutant superoxide dismutase (mSOD), and wt littermates. We find that most GFP+ and CX(3)CR1(+/GFP) cells are found adjacent to the microvasculature within the CNS, both in mSOD and wt mice. GFP+ and CX(3)CR1(+/GFP) cells within the CNS have a variety of morphologies, including cells with an elongated appearance, weak Iba-1 immunoreactivity, and often mannose receptor immunoreactivity, indicating that these cells are perivascular microglia. Typically, less than 10% of BM-derived cells had a stellate-shape and expressed strong Iba-1 immunoreactivity, as expected for parenchymal microglia, indicating that BM-derived cells uncommonly generate parenchymal microglia. Adoptive transfer of BM-derived cells from CX(3)CR1(+/GFP) mice revealed that many elongated cells are GFP+, demonstrating that some perivascular cells are derived from BM cells of the CX(3)CR1+ lineage. The significantly greater numbers of BM cells in mSOD than in control mice indicate that the presence of these BM cells in the spinal cord is regulated by conditioning stimuli that may include irradiation and inflammatory factors within the CNS.

Neuromuscular dysfunction in the mutant superoxide dismutase mouse model of amyotrophic lateral sclerosis.

To better understand the interaction between motor neuron dysfunction and denervation in amyotrophic lateral sclerosis (ALS), we have evaluated motor neuron number and the retrograde uptake and transport of fluorogold by motor neurons in mice overexpressing mutant superoxide dismutase (mSOD), and wild-type controls. N-CAM immunoreactivity and protein kinase expression were determined in skeletal muscle during denervation. We found that in severely affected mSOD mice, motor neuron loss is moderate (approximate 40% reduction), whereas retrograde uptake/transport as assessed using fluorogold is profoundly impaired (approximately 90% reduction). The impairment in fluorogold uptake/transport corresponds to measures of progressive muscle denervation such as increased N-CAM immunoreactivity of muscle and increased expression of protein kinase B (PKB) in denervated muscle. These data suggest that the debility in the mSOD mouse model of ALS is produced, in part, by impaired retrograde uptake/transport in motor neuron axons in spite of regenerative support from muscle such as elevated expression of PKB.

Bone Marrow-Derived Cell Accumulation in the Spinal Cord Is Independent of Peripheral Mobilization in a Mouse Model of Amyotrophic Lateral Sclerosis.

Bone marrow-derived cells (BMDCs) are capable of migrating across the blood-brain barrier (BBB) and accumulating in the central nervous system (CNS) when transplanted into recipients conditioned with whole-body irradiation or chemotherapy. We used the chemotherapeutic agents busulfan and treosulfan to condition recipient mice for transplantation with bone marrow (BM) cells isolated from donor mice ubiquitously expressing green fluorescent protein. We attempted to increase the accumulation of BMDCs in the CNS by mobilization of BMDCs using either, or both, granulocyte colony-stimulating factor (GCSF) or plerixafor (AMD3100). We also used several concentrations of busulfan. We hypothesized that higher concentrations of busulfan and BMDC mobilization would increase numbers of GFP+ cells in the CNS. The doses of busulfan employed (60-125 mg/kg) all resulted in high levels of sustained chimerism (>85% 1 year post-transplant) in both the blood and BM of wild-type (WT) mice and an amyotrophic lateral sclerosis (ALS) mouse model. Moreover, cells accumulated within the CNS in a dose-, time-, and disease-dependent manner. Conditioning with the hydrophilic busulfan analog treosulfan, which is unable to cross the BBB efficiently, also resulted in a high degree of BM chimerism. However, few GFP+ BMDCs were found within the CNS of WT or ALS mice of treosulfan-conditioned mice. Mobilization of BMDCs into the circulation using GCSF and/or AMD3100 did not lead to increased accumulation of GFP+ BMDCs within the CNS of WT or ALS mice. Weekly analysis of BMDC accumulation revealed that BMDCs accumulated more rapidly and to a greater extent in the CNS of ALS mice conditioned with a high dose (125 mg/kg) of busulfan compared to a lower dose (80 mg/kg). The number of GFP+ BMDCs in the CNS labeling with the proliferation marker Ki67 increased in parallel with BMDC accumulation within the CNS. Our results indicate that establishment of high levels of blood and BM chimerism alone is not sufficient to induce BMDC accumulation within the CNS and that CNS conditioning is a crucial requirement for BMDC accumulation to occur. Moreover, it appears that proliferation of BMDCs that infiltrate the CNS is partly responsible for cell accumulation in busulfan-conditioned ALS mice.

Clinical and neuropathological features of ALS/FTD with TIA1 mutations.

Mutations in the stress granule protein T-cell restricted intracellular antigen 1 (TIA1) were recently shown to cause amyotrophic lateral sclerosis (ALS) with or without frontotemporal dementia (FTD). Here, we provide detailed clinical and neuropathological descriptions of nine cases with TIA1 mutations, together with comparisons to sporadic ALS (sALS) and ALS due to repeat expansions in C9orf72 (C9orf72+). All nine patients with confirmed mutations in TIA1 were female. The clinical phenotype was heterogeneous with a range in the age at onset from late twenties to the eighth decade (mean = 60 years) and disease duration from one to 6 years (mean = 3 years). Initial presentation was either focal weakness or language impairment. All affected individuals received a final diagnosis of ALS with or without FTD. No psychosis or parkinsonism was described. Neuropathological examination on five patients found typical features of ALS and frontotemporal lobar degeneration (FTLD-TDP, type B) with anatomically widespread TDP-43 proteinopathy. In contrast to C9orf72+ cases, caudate atrophy and hippocampal sclerosis were not prominent. Detailed evaluation of the pyramidal motor system found a similar degree of neurodegeneration and TDP-43 pathology as in sALS and C9orf72+ cases; however, cases with TIA1 mutations had increased numbers of lower motor neurons containing round eosinophilic and Lewy body-like inclusions on HE stain and round compact cytoplasmic inclusions with TDP-43 immunohistochemistry. Immunohistochemistry and immunofluorescence failed to demonstrate any labeling of inclusions with antibodies against TIA1. In summary, our TIA1 mutation carriers developed ALS with or without FTD, with a wide range in age at onset, but without other neurological or psychiatric features. The neuropathology was characterized by widespread TDP-43 pathology, but a more restricted pattern of neurodegeneration than C9orf72+ cases. Increased numbers of round eosinophilic and Lewy-body like inclusions in lower motor neurons may be a distinctive feature of ALS caused by TIA1 mutations.

TIA1 Mutations in Amyotrophic Lateral Sclerosis and Frontotemporal Dementia Promote Phase Separation and Alter Stress Granule Dynamics.

Amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) are age-related neurodegenerative disorders with shared genetic etiologies and overlapping clinical and pathological features. Here we studied a novel ALS/FTD family and identified the P362L mutation in the low-complexity domain (LCD) of T cell-restricted intracellular antigen-1 (TIA1). Subsequent genetic association analyses showed an increased burden of TIA1 LCD mutations in ALS patients compared to controls (p = 8.7 × 10-6). Postmortem neuropathology of five TIA1 mutations carriers showed a consistent pathological signature with numerous round, hyaline, TAR DNA-binding protein 43 (TDP-43)-positive inclusions. TIA1 mutations significantly increased the propensity of TIA1 protein to undergo phase transition. In live cells, TIA1 mutations delayed stress granule (SG) disassembly and promoted the accumulation of non-dynamic SGs that harbored TDP-43. Moreover, TDP-43 in SGs became less mobile and insoluble. The identification of TIA1 mutations in ALS/FTD reinforces the importance of RNA metabolism and SG dynamics in ALS/FTD pathogenesis.

Adducin at the Neuromuscular Junction in Amyotrophic Lateral Sclerosis: Hanging on for Dear Life.

The neurological dysfunction in amyotrophic lateral sclerosis (ALS)/motor neurone disease (MND) is associated with defective nerve-muscle contacts early in the disease suggesting that perturbations of cell adhesion molecules (CAMs) linking the pre- and post-synaptic components of the neuromuscular junction (NMJ) are involved. To search for candidate proteins implicated in this degenerative process, researchers have studied the Drosophila larval NMJ and find that the cytoskeleton-associated protein, adducin, is ideally placed to regulate synaptic contacts. By controlling the levels of synaptic proteins, adducin can de-stabilize synaptic contacts. Interestingly, elevated levels of phosphorylated adducin have been reported in ALS patients and in a mouse model of the disease. Adducin is regulated by phosphorylation through protein kinase C (PKC), some isoforms of which exhibit Ca(2+)-dependence, raising the possibility that changes in intracellular Ca(2+) might alter PKC activation and secondarily influence adducin phosphorylation. Furthermore, adducin has interactions with the alpha subunit of the Na(+)/K(+)-ATPase. Thus, the phosphorylation of adducin may secondarily influence synaptic stability at the NMJ and so influence pre- and post-synaptic integrity at the NMJ in ALS.

Submyeloablative conditioning with busulfan permits bone marrow-derived cell accumulation in a murine model of Alzheimer's disease.

Previous work has suggested that bone marrow (BM)-derived cells (BMDCs) accumulate within the CNS and could potentially associate with ß-amyloid plaques in Alzheimer's disease (AD). To explore the accumulation of BMDCs in murine AD, we transplanted green fluorescent protein (GFP)-labeled BM cells into triple transgenic (3×Tg) and wild-type (wt) mice using non-irradiative myelosuppresive conditioning with busulfan (BU). We find that BU (80mg/kg) is sufficient to obtain adequate chimerism (>85%) in wt mice. In order to obtain appreciable non-irradiative chimerism in the 3×Tg mice (>80%), anti-asialo ganglio-N-tetraosylceramide (α-ASGM-1) antibody was also used to reduce natural killer cell function and thereby abrogate the hybrid resistance of the 3×Tg mouse strain. Using BU conditioning and α-ASGM-1 together, we observed sustained BM chimerism and BMDC accumulation within the CNS of the 3×Tg and wt mice. In cortex and hippocampus, BMDC accumulation was perivascular in distribution and similar between 3×Tg and wt mice, with no clear association between BMDCs and AD plaques. We conclude that non-irradiative BM chimerism can be achieved with BU in 3×Tg mice, but requires α-ASGM-1 (or similar appropriate NK-cell depletion). Use of this chimerism protocol permits BMDCs accumulation in the CNS of mixed strain recipient mice although BMDCs appear to be largely perivascular within cortex and hippocampus.

Busulfan as a myelosuppressive agent for generating stable high-level bone marrow chimerism in mice.

Bone marrow transplantation (BMT) is often used to replace the bone marrow (BM) compartment of recipient mice with BM cells expressing a distinct biomarker isolated from donor mice. This technique allows for identification of donor-derived hematopoietic cells within the recipient mice, and can be used to isolate and characterize donor cells using various biochemical techniques. BMT typically relies on myeloablative conditioning with total body irradiation to generate niche space within the BM compartment of recipient mice for donor cell engraftment. The protocol we describe here uses myelosuppressive conditioning with the chemotherapeutic agent busulfan. Unlike irradiation, which requires the use of specialized facilities, busulfan conditioning is performed using intraperitoneal injections of 20 mg/kg busulfan until a total dose of 60-100 mg/kg has been administered. Moreover, myeloablative irradiation can have toxic side effects and requires successful engraftment of donor cells for survival of recipient mice. In contrast, busulfan conditioning using these doses is generally well tolerated and mice survive without donor cell support. Donor BM cells are isolated from the femurs and tibiae of mice ubiquitously expressing green fluorescent protein (GFP), and injected into the lateral tail vein of conditioned recipient mice. BM chimerism is estimated by quantifying the number of GFP+ cells within the peripheral blood following BMT. Levels of chimerism >80% are typically observed in the peripheral blood 3-4 weeks post-transplant and remain established for at least 1 year. As with irradiation, conditioning with busulfan and BMT allows for the accumulation of donor BM-derived cells within the central nervous system (CNS), particularly in mouse models of neurodegeneration. This busulfan-mediated CNS accumulation may be more physiological than total body irradiation, as the busulfan treatment is less toxic and CNS inflammation appears to be less extensive. We hypothesize that these cells can be genetically engineered to deliver therapeutics to the CNS.

Detection of in situ protein-protein complexes at the Drosophila larval neuromuscular junction using proximity ligation assay.

Discs large (Dlg) is a conserved member of the membrane-associated guanylate kinase family, and serves as a major scaffolding protein at the larval neuromuscular junction (NMJ) in Drosophila. Previous studies have shown that the postsynaptic distribution of Dlg at the larval NMJ overlaps with that of Hu-li tai shao (Hts), a homologue to the mammalian adducins. In addition, Dlg and Hts are observed to form a complex with each other based on co-immunoprecipitation experiments involving whole adult fly lysates. Due to the nature of these experiments, however, it was unknown whether this complex exists specifically at the NMJ during larval development. Proximity Ligation Assay (PLA) is a recently developed technique used mostly in cell and tissue culture that can detect protein-protein interactions in situ. In this assay, samples are incubated with primary antibodies against the two proteins of interest using standard immunohistochemical procedures. The primary antibodies are then detected with a specially designed pair of oligonucleotide-conjugated secondary antibodies, termed PLA probes, which can be used to generate a signal only when the two probes have bound in close proximity to each other. Thus, proteins that are in a complex can be visualized. Here, it is demonstrated how PLA can be used to detect in situ protein-protein interactions at the Drosophila larval NMJ. The technique is performed on larval body wall muscle preparations to show that a complex between Dlg and Hts does indeed exist at the postsynaptic region of NMJs.