Mice Transgenic for the Human Carcinoembryonic Antigen Gene Maintain Its Spatiotemporal Expression Pattern

The tumor marker carcinoembryonic antigen (CEA) is predominantly expressed in epithelial cells along the gastrointestinal tract and in a variety of adenocarcinomas. As a basis for investigating its in vivo regulation and for establishing an animal model for tumor immunotherapy, transgenic mice were generated with a 33-kilobase cosmid clone insert containing the complete human CEA gene and flanking sequences. CEA was found in the tongue, esophagus, stomach, small intestine, cecum, colon, and trachea and at low levels in the lung, testis, and uterus of adult mice of independent transgenic strains. CEA was first detected at day 10.5 of embryonic development (embryonic day 10.5) in primary trophoblast giant cells and was found in the developing gut, urethra, trachea, lung, and nucleus pulposus of the vertebral column from embryonic day 14.5 onwards. From embryonic day 16.5 CEA was also visible in the nasal mucosa and tongue. Because this spatiotemporal expression pattern correlates well with that known for humans, it follows that the transferred genomic region contains all of the regulatory elements required for the correct expression of CEA. Furthermore, although mice apparently lack an endogenous CEA gene, the entire repertoire of transcription factors necessary for correct expression of the CEA transgene is conserved between mice and humans. After tumor induction, these immunocompetent mice will serve as a model for optimizing various forms of immunotherapy, using CEA as a target antigen

Neuropathology in Mice Expressing Mouse Alpha-Synuclein

α-Synuclein (αSN) in human is tightly linked both neuropathologically and genetically to Parkinson's disease (PD) and related disorders. Disease-causing properties in vivo of the wildtype mouse ortholog (mαSN), which carries a threonine at position 53 like the A53T human mutant version that is genetically linked to PD, were never reported. To this end we generated mouse lines that express mαSN in central neurons at levels reaching up to six-fold compared to endogenous mαSN. Unlike transgenic mice expressing human wildtype or mutant forms of αSN, these mαSN transgenic mice showed pronounced ubiquitin immunopathology in spinal cord and brainstem. Isoelectric separation of mαSN species revealed multiple isoforms including two Ser129-phosphorylated species in the most severely affected brain regions. Neuronal Ser129-phosphorylated αSN occured in granular and small fibrillar aggregates and pathological staining patterns in neurites occasionally revealed a striking ladder of small alternating segments staining either for Ser129-phosphorylated αSN or ubiquitin but not both. Axonal degeneration in long white matter tracts of the spinal cord, with breakdown of myelin sheaths and degeneration of neuromuscular junctions with loss of integrity of the presynaptic neurofilament network in mαSN transgenic mice, was similar to what we have reported for mice expressing human αSN wildtype or mutant forms. In hippocampal neurons, the mαSN protein accumulated and was phosphorylated but these neurons showed no ubiquitin immunopathology. In contrast to the early-onset motor abnormalities and muscle weakness observed in mice expressing human αSN, mαSN transgenic mice displayed only end-stage phenotypic alterations that manifested alongside with neuropathology. Altogether these findings show that increased levels of wildtype mαSN does not induce early-onset behavior changes, but drives end-stage pathophysiological changes in murine neurons that are strikingly similar to those evoked by expression of human wildtype or mutant forms

The HSP70 Molecular Chaperone Is Not Beneficial in a Mouse Model of α-synucleinopathy

BACKGROUND: Aggregation and misfolded alpha-synuclein is thought to be central in the pathogenesis of Parkinson's disease (PD). Heat-shock proteins (HSPs) that are involved in refolding and degradation processes could lower the aggregate load of alpha-synuclein and thus be beneficial in alpha-synucleinopathies. METHODOLOGY/PRINCIPAL FINDINGS: We co-overexpressed human A53T point-mutated alpha-synuclein and human HSP70 in mice, both under the control of Thy1 regulatory sequences. Behavior read-outs showed no beneficial effect of HSP70 expression in mice. In contrast, motor coordination, grip strength and weight were even worse in the alpha-synucleinopathy model in the presence of HSP70 overexpression. Biochemical analyses revealed no differences in alpha-synuclein oligomers/aggregates, truncations and phosphorylation levels and alpha-synuclein localization was unchanged in immunostainings. CONCLUSION/SIGNIFICANCE: Overexpressing HSP70 in a mouse model of alpha-synucleinopathy did not lower the toxic load of alpha-synuclein species and had no beneficial effect on alpha-synuclein-related motor deficits

γ-Synucleinopathy: neurodegeneration associated with overexpression of the mouse protein

The role of α-synuclein in pathogenesis of familial and idiopathic forms of Parkinson’s disease, and other human disorders known as α-synucleinopathies, is well established. In contrast, the involvement of two other members of the synuclein family, β-synuclein and γ-synuclein, in the development and progression of neurodegeneration is poorly studied. However, there is a growing body of evidence that α-synuclein and β-synuclein have opposite neuropathophysiological effects. Unlike α-synuclein, overexpressed β-synuclein does not cause pathological changes in the nervous system of transgenic mice and even ameliorates the pathology caused by overexpressed α-synuclein. To assess the consequences of excess expression of the third family member, γ-synuclein, on the nervous system we generated transgenic mice expressing high levels of mouse γ-synuclein under control of Thy-1 promoter. These animals develop severe age- and transgene dose-dependent neuropathology, motor deficits and die prematurely. Histopathological changes include aggregation of γ-synuclein, accumulation of various inclusions in neuronal cell bodies and processes, and astrogliosis. These changes are seen throughout the nervous system but are most prominent in the spinal cord where they lead to loss of spinal motor neurons. Our data suggest that down-regulation of small heat shock protein HSPB1 and disintegration of neurofilament network play a role in motor neurons dysfunction and death. These findings demonstrate that γ-synuclein can be involved in neuropathophysiological changes and the death of susceptible neurons suggesting the necessity of further investigations of the potential role of this synuclein in disease

High LRRK2 Levels Fail to Induce or Exacerbate Neuronal Alpha-Synucleinopathy in Mouse Brain

The G2019S mutation in the multidomain protein leucine-rich repeat kinase 2 (LRRK2) is one of the most frequently identified genetic causes of Parkinson’s disease (PD). Clinically, LRRK2(G2019S) carriers with PD and idiopathic PD patients have a very similar disease with brainstem and cortical Lewy pathology (α-synucleinopathy) as histopathological hallmarks. Some patients have Tau pathology. Enhanced kinase function of the LRRK2(G2019S) mutant protein is a prime suspect mechanism for carriers to develop PD but observations in LRRK2 knock-out, G2019S knock-in and kinase-dead mutant mice suggest that LRRK2 steady-state abundance of the protein also plays a determining role. One critical question concerning the molecular pathogenesis in LRRK2(G2019S) PD patients is whether α-synuclein (aSN) has a contributory role. To this end we generated mice with high expression of either wildtype or G2019S mutant LRRK2 in brainstem and cortical neurons. High levels of these LRRK2 variants left endogenous aSN and Tau levels unaltered and did not exacerbate or otherwise modify α-synucleinopathy in mice that co-expressed high levels of LRRK2 and aSN in brain neurons. On the contrary, in some lines high LRRK2 levels improved motor skills in the presence and absence of aSN-transgene-induced disease. Therefore, in many neurons high LRRK2 levels are well tolerated and not sufficient to drive or exacerbate neuronal α-synucleinopathy

LRRK2 protein levels are determined by kinase function and are crucial for kidney and lung homeostasis in mice

Mutations in leucine-rich repeat kinase 2 (LRRK2) cause late-onset Parkinson's disease (PD), but the underlying pathophysiological mechanisms and the normal function of this large multidomain protein remain speculative. To address the role of this protein in vivo, we generated three different LRRK2 mutant mouse lines. Mice completely lacking the LRRK2 protein (knock-out, KO) showed an early-onset (age 6 weeks) marked increase in number and size of secondary lysosomes in kidney proximal tubule cells and lamellar bodies in lung type II cells. Mice expressing a LRRK2 kinase-dead (KD) mutant from the endogenous locus displayed similar early-onset pathophysiological changes in kidney but not lung. KD mutants had dramatically reduced full-length LRRK2 protein levels in the kidney and this genetic effect was mimicked pharmacologically in wild-type mice treated with a LRRK2-selective kinase inhibitor. Knock-in (KI) mice expressing the G2019S PD-associated mutation that increases LRRK2 kinase activity showed none of the LRRK2 protein level and histopathological changes observed in KD and KO mice. The autophagy marker LC3 remained unchanged but kidney mTOR and TCS2 protein levels decreased in KD and increased in KO and KI mice. Unexpectedly, KO and KI mice suffered from diastolic hypertension opposed to normal blood pressure in KD mice. Our findings demonstrate a role for LRRK2 in kidney and lung physiology and further show that LRRK2 kinase function affects LRRK2 protein steady-state levels thereby altering putative scaffold/GTPase activity. These novel aspects of peripheral LRRK2 biology critically impact ongoing attempts to develop LRRK2 selective kinase inhibitors as therapeutics for PD

Gastrin-Releasing Peptide Signaling Plays a Limited and Subtle Role in Amygdala Physiology and Aversive Memory

Links between synaptic plasticity in the lateral amygdala (LA) and Pavlovian fear learning are well established. Neuropeptides including gastrin-releasing peptide (GRP) can modulate LA function. GRP increases inhibition in the LA and mice lacking the GRP receptor (GRPR KO) show more pronounced and persistent fear after single-trial associative learning. Here, we confirmed these initial findings and examined whether they extrapolate to more aspects of amygdala physiology and to other forms of aversive associative learning. GRP application in brain slices from wildtype but not GRPR KO mice increased spontaneous inhibitory activity in LA pyramidal neurons. In amygdala slices from GRPR KO mice, GRP did not increase inhibitory activity. In comparison to wildtype, short- but not long-term plasticity was increased in the cortico-lateral amygdala (LA) pathway of GRPR KO amygdala slices, whereas no changes were detected in the thalamo-LA pathway. In addition, GRPR KO mice showed enhanced fear evoked by single-trial conditioning and reduced spontaneous firing of neurons in the central nucleus of the amygdala (CeA). Altogether, these results are consistent with a potentially important modulatory role of GRP/GRPR signaling in the amygdala. However, administration of GRP or the GRPR antagonist (D-Phe6, Leu-NHEt13, des-Met14)-Bombesin (6–14) did not affect amygdala LTP in brain slices, nor did they affect the expression of conditioned fear following intra-amygdala administration. GRPR KO mice also failed to show differences in fear expression and extinction after multiple-trial fear conditioning, and there were no differences in conditioned taste aversion or gustatory neophobia. Collectively, our data indicate that GRP/GRPR signaling modulates amygdala physiology in a paradigm-specific fashion that likely is insufficient to generate therapeutic effects across amygdala-dependent disorders

Opportunities and challenges for molecular chaperone modulation to treat protein-conformational brain diseases

A common pathological hallmark of protein-conformational brain diseases is the formation of disease-specific protein aggregates. In Alzheimer’s disease (AD), these are comprised of Amyloid-β (Aβ) and Tau as opposed to α-synuclein in Parkinson`s disease (PD) and N-terminal fragments of mutant huntingtin (mHTT) in Huntington’s disease (HD). Most aggregates also sequester molecular chaperones, a protein family that assist in folding, re-folding, stabilization and processing of client proteins including misfolded proteins in brain diseases. Molecular chaperone modulation has achieved remarkable therapeutic effects in some cellular and preclinical animal models of protein-conformational diseases. This has raised hope for chaperone-based strategies to combat these diseases. Here, we briefly review the functional diversity and medical significance of molecular chaperones, their therapeutic potential and common and specific challenges towards clinical application