Masato Hosokawa

Prion-like spreading of pathological α-synuclein in brain

α-Synuclein is the major component of filamentous inclusions that constitute the defining characteristic of neurodegenerative α-synucleinopathies. However, the molecular mechanisms underlying α-synuclein accumulation and spread are unclear. Here we show that intracerebral injections of sarkosyl-insoluble α-synuclein from brains of patients with dementia with Lewy bodies induced hyperphosphorylated α-synuclein pathology in wild-type mice. Furthermore, injection of fibrils of recombinant human and mouse α-synuclein efficiently induced similar α-synuclein pathologies in wild-type mice. C57BL/6J mice injected with α-synuclein fibrils developed abundant Lewy body/Lewy neurite-like pathology, whereas mice injected with soluble α-synuclein did not. Immunoblot analysis demonstrated that endogenous mouse α-synuclein started to accumulate 3 months after inoculation, while injected human α-synuclein fibrils disappeared in about a week. These results indicate that α-synuclein fibrils have prion-like properties and inoculation into wild-type brain induces α-synuclein pathology in vivo. This is a new mouse model of sporadic α-synucleinopathy and should be useful for elucidating progression mechanisms and evaluating disease-modifying therapy.

Transgenic mice overexpressing amyloid beta protein are an incomplete model of Alzheimer disease.

We compared lesions in elderly transgenic (tg) mice carrying the Swedish double mutation KM670/671NL with lesions in Alzheimer disease (AD) by histochemical and immunohistochemical techniques. Highly similar staining for beta-amyloid protein (Abeta) was observed in AD and the mouse models. The abundant amyloid deposits in tg mice were in a consolidated state as revealed by strong Congo red birefringence. In both tg mice and AD, amyloid deposits were ApoE-positive and were surrounded by activated astrocytes. However, Bielschowsky silver staining and immunostaining with tau antibodies revealed no neurofibrillary tangles (NFTs) in the mice as opposed to abundant NFTs in AD. The microglial pattern was also distinctly different. Tg mice had only weakly activated microglia, which expressed low levels of the complement receptor CD11b. They were gathered around the periphery of the deposits. In contrast, AD lesions had strongly activated microglia, which expressed high levels of CD11b. They were associated with the plaque core. Immunostaining for complement proteins was weak in tg mice but very strong in AD deposits. We conclude that the weak inflammatory response and absence of NFTs indicate that tg mice are only a limited model of AD. Therapeutic strategies for the treatment of AD based on tg mouse models that overexpress Abeta may be limited in their application.

Pathological alpha-synuclein propagates through neural networks

Backgroundα-Synuclein is the major component of filamentous inclusions that constitute the defining characteristic of Parkinson’s disease, dementia with Lewy bodies and multiple system atrophy, so-called α-synucleinopathies. Recent studies revealed that intracerebral injection of recombinant α-synuclein fibrils into wild-type mouse brains induced prion-like propagation of hyperphosphorylated α-synuclein pathology. However, the propagation mechanisms of α-synuclein have not been fully elucidated.ResultsIn this study, in order to establish where and how α-synuclein pathology propagates, we injected recombinant mouse α-synuclein fibrils into three different brain areas (substantia nigra, striatum, and entorhinal cortex) of wild-type mice and compared the resulting distributions of α-synuclein pathology at 1 month after injection. Distinct patterns of pathology were observed in mice injected at the different sites. Within one month after injection, the pathology had spread to neurons in areas far from the injection sites, especially areas with direct neural connections to the injection sites. Surprisingly, phosphorylated tau and TDP-43 pathologies were also observed in mice injected with α-synuclein fibrils into striatum and entorhinal cortex at one month after injection. Phosphorylated tau and TDP-43 were accumulated in dot-like inclusions, but these were rarely colocalized with α-synuclein pathology. It seems that accumulation of α-synuclein has a synergistic effect on tau and TDP-43 aggregation. Additionally, intracerebral injection with sarkosyl-insoluble fraction prepared from wild-type mice injected synthetic α-synuclein fibrils can also induce phosphorylated α-synuclein pathology in wild-type mice.ConclusionsOur data indicate that α-synuclein aggregation spread by prion-like mechanisms through neural networks in mouse brains.

Phosphorylated and cleaved TDP-43 in ALS, FTLD and other neurodegenerative disorders and in cellular models of TDP-43 proteinopathy.

Transactivation response (TAR) DNA‐binding protein of Mr 43 kDa (TDP‐43) is a major component of the tau‐negative and ubiquitin‐positive inclusions that characterize amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration which is now referred to as FTLD‐TDP. Concurrent TDP‐43 pathology has been reported in a variety of other neurodegenerative disorders such as Alzheimers disease, forming a group of TDP‐43 proteinopathy. Accumulated TDP‐43 is characterized by phosphorylation and fragmentation. There is a close relationship between the pathological subtypes of FTLD‐TDP and the immunoblot pattern of the C‐terminal fragments of phosphorylated TDP‐43. These results suggest that proteolytic processing of accumulated TDP‐43 may play an important role for the pathological process. In cultured cells, transfected C‐terminal fragments of TDP‐43 are more prone to form aggregates than full‐length TDP‐43. Transfecting the C‐terminal fragment of TDP‐43 harboring pathogenic mutations of TDP‐43 gene identified in familial and sporadic ALS cases into cells enhanced the aggregate formation. Furthermore, we found that methylene blue and dimebon inhibit aggregation of TDP‐43 in these cellular models. Understanding the mechanism of phosphorylation and truncation of TDP‐43 and aggregate formation may be crucial for clarifying the pathogenesis of TDP‐43 proteinopathy and for developing useful therapeutics.

Expression of complement messenger RNAs and proteins by human oligodendroglial cells.

Neurons, astrocytes, microglia, and endothelial cells are capable of synthesizing most, if not all, of the complement proteins. Little is known, however, about the capacity of oligodendroglial cells to generate complement components. This study evaluated expression of complement mRNAs and their protein products by human oligodendrocytes. Cells were isolated and cultured from white matter of seven adult cases that had undergone surgical temporal lobe resection for epilepsy. Oligodendroglial cultures were characterized by the expression of such cell type‐specific mRNAs as myelin proteolipid protein (PLP), oligodendrocyte‐specific protein (OSP), and 2′,3′‐cyclic nucleotide 3′‐phosphodiesterase (CNPase) and were further characterized by immunostaining for such differentiation markers as myelin basic protein (MBP), PLP, CNPase, and O4. RT‐PCR analysis showed that the oligodendroglial cells expressed detectable levels of complement mRNAs for the C1q B‐chain, C1r, C1s, C2, C3, C4, C5, C6, C7, C8 γ subunit, and C9. Immunostaining was positive for C1q, C1s, C2, C3, C4, C5, C6, C7, C8, and C9. Double immunostaining for the oligodendrocyte marker O4 and the complement protein C3 demonstrated that all O4‐positive cells were also positive for C3, indicating constitutive C3 expression. These results indicate that oligodendroglial cells may be a source of complement proteins in human brain and thus could contribute to the pathogenesis of several neurodegenerative and inflammatory diseases of the CNS, such as Alzheimers disease, multiple sclerosis, and progressive supranuclear palsy, where complement‐activated oligodendrocytes are abundant. GLIA 42:417–423, 2003.

A chemical chaperone, sodium 4-phenylbutyric acid, attenuates the pathogenic potency in human α-synuclein A30P + A53T transgenic mice

Aggregation and cytotoxicity of misfolded alpha-synuclein are postulated to be crucial in the disease processes of Parkinsons disease (PD) and other synucleinopathies. Mutations in the alpha-synuclein gene in some pedigrees of familial PD have been reported. The mutant alpha-synuclein has been reported to form fibrillar aggregates resulting in biochemical abnormalities that are responsible for the onset of familial PD. Thus, any agent that effectively prevents the development of misfolded and aggregated alpha-synuclein would be a disease modifying therapeutic candidate. We examined the efficacy of sodium 4-phenylbutyric acid (PBA), one of the chemical chaperons, in transgenic (Tg) mice overexpressing human alpha-synuclein containing a double mutation (A30P + A53T). To evaluate the therapeutic efficacy, bradykinesia and motor coordination were assessed using a pole test and a rotarod treadmill task, respectively. After PBA treatment, these motor deteriorations gradually improved. In immunohistochemical examinations, both a loss of tyrosine hydroxylase-positive neurons and an increase of phosphorylated alpha-synuclein in the substantia nigra were inhibited, resulting in no depletion of the striatal dopamine content. These data suggest that PBA might be one of the therapeutic reagents for neurodegenerative disorders.

Methylene Blue Reduced Abnormal Tau Accumulation in P301L Tau Transgenic Mice

In neurodegenerative disorders, abnormally hyperphosphorylated and aggregated tau accumulates intracellularly, a mechanism which is thought to induce neuronal cell death. Methylene blue, a type of phenothiazine, has been reported to inhibit tau aggregation in vitro. However, the effect of methylene blue in vivo has remained unknown. Therefore, we examined whether methylene blue suppresses abnormal tau accumulation using P301L tau transgenic mice. At 8 to 11 months of age, these mice were orally administered methylene blue for 5 months. Subsequent results of Western blotting analysis revealed that this agent reduced detergent-insoluble phospho-tau. Methylene blue may have potential as a drug candidate for the treatment of tauopathy.

Molecular analysis and biochemical classification of TDP-43 proteinopathy

Amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TAR DNA-binding protein of 43 kDa pathology are progressive neurodegenerative diseases that are characterized by intracytoplasmic aggregates of hyperphosphorylated TAR DNA-binding protein of 43 kDa. These TAR DNA-binding protein 43 proteinopathies can be classified into subtypes, which are closely correlated with clinicopathological phenotypes, although the differences in the molecular species of TAR DNA-binding protein 43 in these diseases and the biological significance thereof, remain to be clarified. Here, we have shown that although the banding patterns of abnormally phosphorylated C-terminal fragments of TAR DNA-binding protein 43 differ between the neuropathological subtypes, these are indistinguishable between multiple brain regions and spinal cord in individual patients. Immunoblot analysis of protease-resistant TAR DNA-binding protein 43 demonstrated that the fragment patterns represent different conformations of TAR DNA-binding protein 43 molecular species in the diseases. These results suggest a new clinicopathological classification of TAR DNA-binding protein 43 proteinopathies based on their molecular properties.

The Src/c-Abl pathway is a potential therapeutic target in amyotrophic lateral sclerosis

Analysis of ALS patient iPSC-derived motor neurons indicates that Src/c-Abl inhibitors may have potential for treating ALS. A stepping stone to ALS drug discovery ALS is a heterogeneous motor neuron disease for which there is no treatment and for which a common therapeutic target has yet to be identified. In a new study, Imamura et al. developed a drug screen using motor neurons generated from ALS patient induced pluripotent stem cells (iPSCs). They screened existing drugs and showed that inhibitors of Src/c-Abl kinases promoted autophagy and rescued ALS motor neurons from degeneration. One of the drugs was effective for promoting survival of motor neurons derived from ALS patients with different genetic mutations. The Src/c-Abl pathway may be a potential therapeutic target for developing new drugs to treat ALS. Amyotrophic lateral sclerosis (ALS), a fatal disease causing progressive loss of motor neurons, still has no effective treatment. We developed a phenotypic screen to repurpose existing drugs using ALS motor neuron survival as readout. Motor neurons were generated from induced pluripotent stem cells (iPSCs) derived from an ALS patient with a mutation in superoxide dismutase 1 (SOD1). Results of the screen showed that more than half of the hits targeted the Src/c-Abl signaling pathway. Src/c-Abl inhibitors increased survival of ALS iPSC-derived motor neurons in vitro. Knockdown of Src or c-Abl with small interfering RNAs (siRNAs) also rescued ALS motor neuron degeneration. One of the hits, bosutinib, boosted autophagy, reduced the amount of misfolded mutant SOD1 protein, and attenuated altered expression of mitochondrial genes. Bosutinib also increased survival in vitro of ALS iPSC-derived motor neurons from patients with sporadic ALS or other forms of familial ALS caused by mutations in TAR DNA binding protein (TDP-43) or repeat expansions in C9orf72. Furthermore, bosutinib treatment modestly extended survival of a mouse model of ALS with an SOD1 mutation, suggesting that Src/c-Abl may be a potentially useful target for developing new drugs to treat ALS.

Moderate maternal food restriction in mice impairs physical growth, behavior, and neurodevelopment of offspring

Intrauterine growth retardation (IUGR) occurs in 3% to 7% of all pregnancies. Recent human studies have indicated that neurodevelopmental disabilities, learning disorders, memory impairment, and mood disturbance are common in IUGR offspring. However, the interactions between IUGR and neurodevelopmental disorders are unclear because of the wide range of causes of IUGR, such as maternal malnutrition, placental insufficiency, pregnancy toxemia, and fetal malformations. Meanwhile, many studies have shown that moderate food restriction enhances spatial learning and improves mood disturbance in adult humans and animals. To date, the effects of maternal moderate food restriction on fetal brain remain largely unknown. In this study, we hypothesized that IUGR would be caused by even moderate food restriction in pregnant females and that the offspring would have neurodevelopmental disabilities. Mid-pregnant mice received moderate food restriction through the early lactation period. The offspring were tested for aspects of physical development, behavior, and neurodevelopment. The results showed that moderate maternal food restriction induced IUGR. Offspring had low birth weight and delayed development of physical and coordinated movement. Moreover, IUGR offspring exhibited mental disabilities such as anxiety and poor cognitive function. In particular, male offspring exhibited significantly impaired cognitive function at 3 weeks of age. These results suggested that a restricted maternal diet could be a risk factor for developmental disability in IUGR offspring and that male offspring might be especially susceptible.