Viviana Cantillana

Loss of tau elicits axonal degeneration in a mouse model of Alzheimer's disease.

A central issue in the pathogenesis of tauopathy is the question of how tau protein dysfunction leads to neurodegeneration. We have previously demonstrated that the absence of tau protein is associated with destabilization of microtubules and impaired neurite outgrowth (Dawson et al., 2001; Rapoport et al., 2002). We now hypothesize that the absence of functional tau protein may render the central nervous system more vulnerable to secondary insults such as the overexpression of mutated beta amyloid precursor protein (APP) and traumatic brain injury. We therefore crossed tau knockout mice (Dawson et al., 2001) to mice overexpressing a mutated human APP (APP(670,671), A(sw)) (Hsiao et al., 1996) and created a mouse model (A(sw)/mTau(-/-)) that provides evidence that the loss of tau function causes degeneration of neuronal processes. The overexpression of APP(670,671) in tau knockout mice, elicits the extensive formation of axonal spheroids. While spheroids are only found associated with Abeta plaques in mice expressing APP(670,671) on an endogenous mouse tau background (Irizarry et al., 1997), A(sw)/mTau(-/-) mice have spheroids not only surrounding Abeta plaques but also in white matter tracks and in the neuropil. Plaque associated and neuropil dystrophic neurites and spheroids are prominent features of Alzheimers disease (Masliah et al., 1993; Terry, 1996; Stokin et al., 2005), and our current data suggests that loss of tau function may lead to neurodegeneration.

NO synthase 2 (NOS2) deletion promotes multiple pathologies in a mouse model of Alzheimer's disease

Alzheimers disease is characterized by two primary pathological features: amyloid plaques and neurofibrillary tangles. The interconnection between amyloid and tau aggregates is of intense interest, but mouse models have yet to reveal a direct interrelationship. We now show that NO may be a key factor that connects amyloid and tau pathologies. Genetic removal of NO synthase 2 in mice expressing mutated amyloid precursor protein results in pathological hyperphosphorylation of mouse tau, its redistribution to the somatodendritic compartment in cortical and hippocampal neurons, and aggregate formation. Lack of NO synthase 2 in the amyloid precursor protein Swedish mutant mouse increased insoluble β-amyloid peptide levels, neuronal degeneration, caspase-3 activation, and tau cleavage, suggesting that NO acts at a junction point between β-amyloid peptides, caspase activation, and tau aggregation.

The Tau N279K Exon 10 Splicing Mutation Recapitulates Frontotemporal Dementia and Parkinsonism Linked to Chromosome 17 Tauopathy in a Mouse Model

Intracellular tau deposits are characteristic of several neurodegenerative disorders called tauopathies. The tau protein regulates the stability and assembly of microtubules by binding to microtubules through three or four microtubule-binding repeats (3R and 4R). The number of microtubule-binding repeats is determined by the inclusion or exclusion of the second microtubule-binding repeat encoded by exon 10 of the TAU gene. TAU gene mutations that alter the inclusion of exon 10, and hence the 4R:3R ratio, are causal in the tauopathy frontotemporal dementia and parkinsonism linked to chromosome 17 (FTDP-17). A mutation located in exon 10 has been identified in several FTDP-17 families that present with increased exon 10 inclusion in both mRNA and protein, parkinsonism, movement disorders, and dementia. We have engineered a human tau minigene construct that was designed to allow alternative splicing of the tau exon 10. Here we demonstrate that transgenic mice expressing human tau protein with this mutation develop neurodegeneration as result of aberrant splicing. The mice recapitulate many of the disease hallmarks that are seen in patients with this mutation, including increased tau exon 10 inclusion in both mRNA and protein, motor and behavioral deficits, and tau protein accumulation in neurons and tufted astrocytes. Furthermore, these mice present with degeneration of the nigrostriatal dopaminergic pathway, suggesting a possible mechanism for parkinsonism in FTDP-17. Additionally, activated caspase-3 immunoreactivity in both neurons and astrocytes implicates the involvement of the apoptotic pathway in the pathology of these mice.

Irgm1-deficient mice exhibit Paneth cell abnormalities and increased susceptibility to acute intestinal inflammation.

Crohns disease (CD) is a chronic, immune-mediated, inflammatory disorder of the intestine that has been linked to numerous susceptibility genes, including the immunity-related GTPase (IRG) M (IRGM). IRGs comprise a family of proteins known to confer resistance to intracellular infections through various mechanisms, including regulation of phagosome processing, cell motility, and autophagy. However, despite its association with CD, the role of IRGM and other IRGs in regulating intestinal inflammation is unclear. We investigated the involvement of Irgm1, an ortholog of IRGM, in the genesis of murine intestinal inflammation. After dextran sodium sulfate exposure, Irgm1-deficient [Irgm1 knockout (KO)] mice showed increased acute inflammation in the colon and ileum, with worsened clinical responses. Marked alterations of Paneth cell location and granule morphology were present in Irgm1 KO mice, even without dextran sodium sulfate exposure, and were associated with impaired mitophagy and autophagy in Irgm1 KO intestinal cells (including Paneth cells). This was manifested by frequent tubular and swollen mitochondria and increased LC3-positive autophagic structures. Interestingly, these LC3-positive structures often contained Paneth cell granules. These results suggest that Irgm1 modulates acute inflammatory responses in the mouse intestine, putatively through the regulation of gut autophagic processes, that may be pivotal for proper Paneth cell functioning.

Immunity-related GTPase M (IRGM) Proteins Influence the Localization of Guanylate-binding Protein 2 (GBP2) by Modulating Macroautophagy

The immunity-related GTPases (IRGs) are a family of proteins induced by interferon-γ that play a crucial role in innate resistance to intracellular pathogens. The M subfamily of IRG proteins (IRGM) plays a profound role in this context, in part because of the ability of its members to regulate the localization and expression of other IRG proteins. We present here evidence that IRGM proteins affect the localization of the guanylate-binding proteins (GBPs), a second family of interferon-induced GTP-binding proteins that also function in innate immunity. Absence of Irgm1 or Irgm3 led to accumulation of Gbp2 in intracellular compartments that were positive for both the macroautophagy (hereafter referred to as autophagy) marker LC3 and the autophagic adapter molecule p62/Sqstm1. Gbp2 was similarly relocalized in cells in which autophagy was impaired because of the absence of Atg5. Both in Atg5- and IRGM-deficient cells, the IRG protein Irga6 relocalized to the same compartments as Gbp2, raising the possibility of a common regulatory mechanism. However, other data indicated that Irga6, but not Gbp2, was ubiquitinated in IRGM-deficient cells. Similarly, coimmunoprecipitation studies indicated that although Irgm3 did interact directly with Irgb6, it did not interact with Gbp2. Collectively, these data suggest that IRGM proteins indirectly modulate the localization of GBPs through a distinct mechanism from that through which they regulate IRG protein localization. Further, these results suggest that a core function of IRGM proteins is to regulate autophagic flux, which influences the localization of GBPs and possibly other factors that instruct cell-autonomous immune resistance.

TT-301 Inhibits Microglial Activation and Improves Outcome after Central Nervous System Injury in Adult Mice

Background: Microglial inhibition may reduce secondary tissue injury and improve functional outcome following acute brain injury. Utilizing clinically relevant murine models of traumatic brain injury and intracerebral hemorrhage, neuroinflammatory responses and functional outcome were examined in the presence of a potential microglial inhibitor, TT-301. Methods: TT-301 or saline was administered following traumatic brain injury or intracerebral hemorrhage, and then for four subsequent days. The effect of TT-301 on neuroinflammatory responses and neuronal viability was assessed, as well as short-term vestibulomotor deficit (Rotorod) and long-term neurocognitive impairment (Morris water maze). Finally differential gene expression profiles of mice treated with TT-301 were compared with those of vehicle. Results: Reduction in F4/80+ staining was demonstrated at 1 and 10 days, but not 28 days, after injury in mice treated with TT-301 (n = 6). These histologic findings were associated with improved neurologic function as assessed by Rotorod, which improved by 52.7% in the treated group by day 7, and Morris water maze latencies, which improved by 232.5% as a function of treatment (n = 12; P < 0.05). Similar benefit was demonstrated following intracerebral hemorrhage, in which treatment with TT-301 was associated with functional neurologic improvement of 39.6% improvement in Rotorod and a reduction in cerebral edema that was independent of hematoma volume (n = 12; P < 0.05). Differential gene expression was evaluated following treatment with TT-301, and hierarchical cluster analysis implicated involvement of the Janus kinase–Signal Transducer and Activator of Transcription pathway after administration of TT-301 (n = 3/group). Conclusions: Modulation of neuroinflammatory responses through TT-301 administration improved histologic and functional parameters in murine models of acute neurologic injury.

Neuroprotective pentapeptide CN-105 is associated with reduced sterile inflammation and improved functional outcomes in a traumatic brain injury murine model

At present, there are no proven pharmacological treatments demonstrated to improve long term functional outcomes following traumatic brain injury(TBI). In the setting of non-penetrating TBI, sterile brain inflammatory responses are associated with the development of cerebral edema, intracranial hypertension, and secondary neuronal injury. There is increasing evidence that endogenous apolipoprotein E(apoE) modifies the neuroinflammatory response through its role in downregulating glial activation, however, the intact apoE holoprotein does not cross the blood-brain barrier due to its size. To address this limitation, we developed a small 5 amino acid apoE mimetic peptide(CN-105) that mimics the polar face of the apoE helical domain involved in receptor interactions. The goal of this study was to investigate the therapeutic potential of CN-105 in a murine model of closed head injury. Treatment with CN-105 was associated with a durable improvement in functional outcomes as assessed by Rotarod and Morris Water Maze and a reduction in positive Fluoro-Jade B stained injured neurons and microglial activation. Administration of CN-105 was also associated with reduction in mRNA expression of a subset of inflammatory and immune-related genes.

Apolipoprotein E mimetic peptide, CN‐105, improves outcomes in ischemic stroke

At present, the absence of a pharmacological neuroprotectant represents an important unmet clinical need in the treatment of ischemic and traumatic brain injury. Recent evidence suggests that administration of apolipoprotein E mimetic therapies represent a viable therapeutic strategy in this setting. We investigate the neuroprotective and anti‐inflammatory properties of the apolipoprotein E mimetic pentapeptide, CN‐105, in a microglial cell line and murine model of ischemic stroke.

Environmental factors regulate Paneth cell phenotype and host susceptibility to intestinal inflammation in Irgm1-deficient mice

ABSTRACT Crohns disease (CD) represents a chronic inflammatory disorder of the intestinal tract. Several susceptibility genes have been linked to CD, though their precise role in the pathogenesis of this disorder remains unclear. Immunity-related GTPase M (IRGM) is an established risk allele in CD. We have shown previously that conventionally raised (CV) mice lacking the IRGM ortholog, Irgm1 exhibit abnormal Paneth cells (PCs) and increased susceptibility to intestinal injury. In the present study, we sought to utilize this model system to determine if environmental conditions impact these phenotypes, as is thought to be the case in human CD. To accomplish this, wild-type and Irgm1−/− mice were rederived into specific pathogen-free (SPF) and germ-free (GF) conditions. We next assessed how these differential housing environments influenced intestinal injury patterns, and epithelial cell morphology and function in wild-type and Irgm1−/− mice. Remarkably, in contrast to CV mice, SPF Irgm1−/− mice showed only a slight increase in susceptibility to dextran sodium sulfate-induced inflammation. SPF Irgm1−/− mice also displayed minimal abnormalities in PC number and morphology, and in antimicrobial peptide expression. Goblet cell numbers and epithelial proliferation were also unaffected by Irgm1 in SPF conditions. No microbial differences were observed between wild-type and Irgm1−/− mice, but gut bacterial communities differed profoundly between CV and SPF mice. Specifically, Helicobacter sequences were significantly increased in CV mice; however, inoculating SPF Irgm1−/− mice with Helicobacter hepaticus was not sufficient to transmit a pro-inflammatory phenotype. In summary, our findings suggest the impact of Irgm1-deficiency on susceptibility to intestinal inflammation and epithelial function is critically dependent on environmental influences. This work establishes the importance of Irgm1−/− mice as a model to elucidate host-environment interactions that regulate mucosal homeostasis and intestinal inflammatory responses. Defining such interactions will be essential for developing novel preventative and therapeutic strategies for human CD. Summary: In this study, we rederived conventionally raised Irgm1-deficient mice into specific pathogen-free and germ-free conditions. We show that these environments determine how Irgm1 regulates Paneth cell function and gut inflammation susceptibility.