Hippocampal neurobiology and function in an aged mouse model of TDP-43 proteinopathy in an APP/PSEN1 background.

Aging is a major risk factor for Alzheimer's disease (AD), the most common cause of dementia worldwide. TDP-43 proteinopathy is reported to be associated with AD pathology is almost 50% of cases. Our exploratory study examined near end-stage (28 months old) mice selectively driving expression of human TDP-43 in the hippocampus and cortex in an APP/PSEN1 background. We hypothesized that hippocampal neuropathology caused by ß-amyloidosis with TDP-43 proteinopathy induced in this model, resembling the pathology seen in AD cases, manifest with changes in resting state functional connectivity. In vivo magnetic resonance imaging and post-mortem histology were performed on four genotypes: wild type, APP/PSEN1, Camk2a/TDP-43, and Camk2a/TDP-43/APP/PSEN1. Our results revealed loss of functional coupling in hippocampus and amygdala that was associated with severe neuronal loss in dentate gyrus of Camk2a/TDP-43/APP/PSEN1 mice compared to APP/PSEN1 and wild type mice. The loss of cells was accompanied by high background of ß-amyloid plaques with sparse phosphorylated TDP-43 pathology. The survival rate was also reduced in Camk2a/TDP-43/APP/PSEN1 mice compared to other groups. This end-of-life study provides exploratory data to reach a better understanding of the role of TDP-43 hippocampal neuropathology in diseases with co-pathologies of TDP-43 proteinopathy and ß-amyloidosis such as AD and limbic predominant age-related TDP-43 encephalopathy (LATE).

Initial gene vector dosing for studying symptomatology of amyotrophic lateral sclerosis in non-human primates.

BACKGROUND: Most amyotrophic lateral sclerosis (ALS) research has focused on mice, but there are distinct differences in the functional neuroanatomy of the corticospinal pathway in primates vs. rodents. A non-human primate model may be more sensitive and more predictive for therapeutic efficacy. METHODS: Rhesus macaques received recombinant adeno-associated virus (AAV9) encoding either the ALS-related pathological protein TDP-43 or a green fluorescent protein (GFP) control by intravenous administration. Motor function and electromyography were assessed over a nine-month expression interval followed by post-mortem analyses. RESULTS: Recombinant TDP-43 or GFP was stably expressed long term. Although the TDP-43 subjects did not manifest severe paralysis and atrophy, there were trends of a partial disease state in the TDP-43 subjects relative to the control. CONCLUSIONS: These data indicate that a higher gene vector dose will likely be necessary for more robust effects, yet augur that a relevant primate model is feasible.

Selective forelimb impairment in rats expressing a pathological TDP-43 25 kDa C-terminal fragment to mimic amyotrophic lateral sclerosis.

Pathological inclusions containing transactive response DNA-binding protein 43 kDa (TDP-43) are common in several neurodegenerative diseases including amyotrophic lateral sclerosis (ALS). TDP-43 normally localizes predominantly to the nucleus, but during disease progression, it mislocalizes to the cytoplasm. We expressed TDP-43 in rats by an adeno-associated virus (AAV9) gene transfer method that transduces neurons throughout the central nervous system (CNS). To mimic the aberrant cytoplasmic TDP-43 found in disease, we expressed a form of TDP-43 with mutations in the nuclear localization signal sequence (TDP-NLS). The TDP-NLS was detected in both the cytoplasm and the nucleus of transduced neurons. Unlike wild-type TDP-43, expression of TDP-NLS did not induce mortality. However, the TDP-NLS induced disease-relevant motor impairments over 24 weeks. We compared the TDP-NLS to a 25 kDa C-terminal proaggregatory fragment of TDP-43 (TDP-25). The clinical phenotype of forelimb impairment was pronounced with the TDP-25 form, supporting a role of this C-terminal fragment in pathogenesis. The results advance previous rodent models by inducing cytoplasmic expression of TDP-43 in the spinal cord, and the non-lethal phenotype enabled long-term study. Approaching a more relevant disease state in an animal model that more closely mimics underlying mechanisms in human disease could unlock our ability to develop therapeutics.

Genetic strategies to study TDP-43 in rodents and to develop preclinical therapeutics for amyotrophic lateral sclerosis.

The neuropathological hallmark of the majority of amyotrophic lateral sclerosis (ALS) and a class of frontotemporal lobar degeneration is ubiquitinated cytoplasmic aggregates composed of transactive response DNA binding protein 43 kDa (TDP-43). Genetic manipulation of TDP-43 in animal models has been used to study the protein's role in pathogenesis. Transgenic rodents for TDP-43 have recapitulated key aspects of ALS such as paralysis, loss of spinal motor neurons and muscle atrophy. Viral vectors are an alternate approach to express pathological proteins in animals. Use of the recombinant adeno-associated virus vector serotype 9 has permitted widespread transgene expression throughout the central nervous system after intravenous administration. Expressing TDP-43 in rats with this method produced a phenotype that was consistent with and similar to TDP-43 transgenic lines. Increased levels of TDP-43 in the nucleus are toxic to neurons and sufficient to produce ALS-like symptoms. Animal models based on TDP-43 will address the relationships between TDP-43 expression levels, pathology, neuronal loss, muscle atrophy, motor function and causative mechanisms of disease. New targets that modify TDP-43 function, or targets from previous ALS models and other models of spinal cord diseases, could be tested for efficacy in the recent rodent models of ALS based on TDP-43. The vector approach could be an important therapeutic channel because the entire spinal cord can be affected from a one-time peripheral administration.

FUS immunogold labeling TEM analysis of the neuronal cytoplasmic inclusions of neuronal intermediate filament inclusion disease: a frontotemporal lobar degeneration with FUS proteinopathy.

Fused in sarcoma (FUS)-immunoreactive neuronal and glial inclusions define a novel molecular pathology called FUS proteinopathy. FUS has been shown to be a component of inclusions of familial amyotrophic lateral sclerosis with FUS mutation and three frontotemporal lobar degeneration entities, including neuronal intermediate filament inclusion disease (NIFID). The pathogenic role of FUS is unknown. In addition to FUS, many neuronal cytoplasmic inclusions (NCI) of NIFID contain aggregates of α-internexin and neurofilament proteins. Herein, we have shown that: (1) FUS becomes relatively insoluble in NIFID and there are no apparent posttranslational modifications, (2) there are no pathogenic abnormalities in the FUS gene in NIFID, and (3) immunoelectron microscopy demonstrates the fine structural localization of FUS in NIFID which has not previously been described. FUS localized to euchromatin, and strongly with paraspeckles, in nuclei, consistent with its RNA/DNA-binding functions. NCI of varying morphologies were observed. Most frequent were the "loosely aggregated cytoplasmic inclusions," 81% of which had moderate or high levels of FUS immunoreactivity. Much rarer "compact cytoplasmic inclusions" and "tangled twine ball inclusions" were FUS-immunoreactive at their granular peripheries, or heavily FUS-positive throughout, respectively. Thus, FUS may aggregate in the cytoplasm and then admix with neuronal intermediate filament accumulations.

Pathogenic cysteine mutations affect progranulin function and production of mature granulins.

Frontotemporal dementia with ubiquitin-positive inclusions (FTLD-U) can be caused by mutations in the progranulin gene (GRN). Progranulin (PGRN) is a cysteine-rich growth factor, which is proteolytically cleaved by elastase to produce several granulins (GRNs). All FTLD-U mutations in GRN characterized to date result in reduced secreted PGRN protein. We recently reported a Spanish family with progressive non-fluent aphasia and dementia in which a novel C521Y mutation segregates with disease. A second cysteine mutation (C139R) has also been reported to be disease specific. Allele-specific mRNA expression assays in brain reveal that the C521Y mutant allele is expressed at similar levels to the wild-type allele. Furthermore, plasma PGRN levels in C521Y carriers are comparable with non-carrier family relatives, suggesting that the mutation does not affect PGRN protein expression and secretion in vivo. Despite normal PGRN levels C521Y and C139R mutant GRNs show reduced neurite growth-stimulating activity in vitro. Further study revealed that these mutations also cause impaired cleavage of PGRN by elastase. Our data suggest that these mutations affect the function of full-length PGRN as well as elastase cleavage of PGRN into GRNs, leading to neurodegeneration.

VCP mutations causing frontotemporal lobar degeneration disrupt localization of TDP-43 and induce cell death.

Frontotemporal lobar degeneration (FTLD) with inclusion body myopathy and Paget disease of bone is a rare, autosomal dominant disorder caused by mutations in the VCP (valosin-containing protein) gene. The disease is characterized neuropathologically by frontal and temporal lobar atrophy, neuron loss and gliosis, and ubiquitin-positive inclusions (FTLD-U), which are distinct from those seen in other sporadic and familial FTLD-U entities. The major component of the ubiquitinated inclusions of FTLD with VCP mutation is TDP-43 (TAR DNA-binding protein of 43 kDa). TDP-43 proteinopathy links sporadic amyotrophic lateral sclerosis, sporadic FTLD-U, and most familial forms of FTLD-U. Understanding the relationship between individual gene defects and pathologic TDP-43 will facilitate the characterization of the mechanisms leading to neurodegeneration. Using cell culture models, we have investigated the role of mutant VCP in intracellular trafficking, proteasomal function, and cell death and demonstrate that mutations in the VCP gene 1) alter localization of TDP-43 between the nucleus and cytosol, 2) decrease proteasome activity, 3) induce endoplasmic reticulum stress, 4) increase markers of apoptosis, and 5) impair cell viability. These results suggest that VCP mutation-induced neurodegeneration is mediated by several mechanisms.

Potentiation of insulin-stimulated glucose transport by the AMP-activated protein kinase.

Data from the use of activators and inhibitors of the AMP-activated protein kinase (AMPK) suggest that AMPK increases sensitivity of glucose transport to stimulation by insulin in muscle cells. We assayed insulin action after adenoviral (Ad) transduction of constitutively active (CA; a truncated form of AMPKalpha(1)) and dominant-negative (DN; which depletes endogenous AMPKalpha) forms of AMPKalpha (Ad-AMPKalpha-CA and Ad-AMPKalpha-DN, respectively) into C(2)C(12) myotubes. Compared with control (Ad-green fluorescent protein), Ad-AMPK-CA increased the ability of insulin to stimulate glucose transport. The increased insulin action in cells expressing AMPK-CA was suppressed by compound C (an AMPK inhibitor). Exposure of cells to 5-aminoimidazole-4-carboxamide-1beta-D-ribofuranoside (an AMPK activator) increased insulin action in uninfected myotubes and myotubes transduced with green fluorescent protein but not in Ad-AMPK-DN-infected myotubes. In Ad-AMPK-CA-transduced cells, serine phosphorylation of insulin receptor substrate 1 was decreased at a mammalian target of rapamycin (or p70 S6 kinase) target site that has been reported to be associated with insulin resistance. These data suggest that, in myotubes, activated AMPKalpha(1) is sufficient to increase insulin action and that the presence of functional AMPKalpha is required for 5-aminoimidazole-4-carboxamide-1beta,D-ribofuranoside-related increases in insulin action.