Alterations in the lipid composition of endosomal-lysosomal membranes may constitute an early event in Alzheimer's disease (AD) pathogenesis. In this study, we investigated the possibility that GM2 ganglioside accumulation in a mouse model of Sandhoff disease might be associated with the accumulation of intraneuronal and extracellular proteins commonly observed in AD. Our results show intraneuronal accumulation of amyloid-β peptide (Aβ)-like, α-synuclein-like, and phospho-tau-like immunoreactivity in the brains of β-hexosaminidase knock-out (HEXB KO) mice. Biochemical and immunohistochemical analyses confirmed that at least some of the intraneuronal Aβ-like immunoreactivity (iAβ-LIR) represents amyloid precursor protein C-terminal fragments (APP-CTFs) and/or Aβ. In addition, we observed increased levels of Aβ40 and Aβ42 peptides in the lipid-associated fraction of HEXB KO mouse brains, and intraneuronal accumulation of ganglioside-bound Aβ (GAβ) immunoreactivity in a brain region-specific manner. Furthermore, α-synuclein and APP-CTFs and/or Aβ were found to accumulate in different regions of the substantia nigra, indicating different mechanisms of accumulation or turnover pathways. Based on the localization of the accumulated iAβ-LIR to endosomes, lysosomes, and autophagosomes, we conclude that a significant accumulation of iAβ-LIR may be associated with the lysosomal-autophagic turnover of Aβ and fragments of APP-containing Aβ epitopes. Importantly, intraneuronal GAβ immunoreactivity, a proposed prefibrillar aggregate found in AD, was found to accumulate throughout the frontal cortices of postmortem human GM1 gangliosidosis, Sandhoff disease, and Tay-Sachs disease brains. Together, these results establish an association between the accumulation of gangliosides, autophagic vacuoles, and the intraneuronal accumulation of proteins associated with AD.

BackgroundReelin and Notch-1 signaling pathways have been recently found to be necessary to induce the expression of brain lipid binding protein (BLBP) and to promote the process extension and the maturation of the neuronal progenitors, the radial glial cells. In this study, we report the cross talk between these two pathways.ResultsBoth in vitro Reelin treatment and overexpression of Notch-1 intracellular domain (NICD) induced BLBP expression and a radial glial phenotype in an immortalized human neural progenitor (HNP) cell line, isolated from the cortex of 14 weeks old fetus. Reelin treatment increased the level of NICD, indicating that Reelin signaling directly activates Notch-1. In addition, reducing NICD release, by inhibiting γ-secretase activity, inhibited the Reelin-induced radial glial phenotype in human neural progenitor cells. Furthermore, we found that Dab-1, an adaptor protein downstream of Reelin, was co-immunoprecipitated with Notch-1 and NICD.ConclusionThese data indicate that Reelin signaling induces BLBP expression and a radial glial phenotype in human neural progenitor cells via the activation of Notch-1. This study suggest that Reelin signaling may act to fine tune Notch-1 activation to favor the induction of a radial glial phenotype prenataly and would thus offer an insight into how Notch-1 signaling leads to different cellular fates at different developmental stages.

DARPP-32 (dopamine and adenosine 3Ј, 5Ј-cyclic monophosphate cAMP-regulated phosphoprotein, 32 kDa) is a striatal-enriched protein that mediates signaling by dopamine and other first messengers in the medium spiny neurons. The transcriptional mechanisms that regulate striatal DARPP-32 expression remain enigmatic and are a subject of much interest in the efforts to induce a striatal phenotype in stem cells. We report the identification and characterization of a conserved region, also known as H10, in intron IV of the gene that codes for DARPP-32 (Ppp1r1b). This DNA sequence forms multiunit complexes with nuclear proteins from adult and embryonic striata of mice and rats. Purification of proteins from these complexes identified early growth response-1 (Egr-1). The interaction between Egr-1 and H10 was confirmed in vitro and in vivo by super-shift and chromatin immunoprecipitation assays, respectively. Importantly, brain-derived neurotrophic factor (BDNF), a known inducer of DARPP-32 and Egr-1 expression, enhanced Egr-1 binding to H10 in vitro. Moreover, overexpression of Egr-1 in primary striatal neurons induced the expression of DARPP-32, whereas a dominantnegative Egr-1 blocked DARPP-32 induction by BDNF. Together, this study identifies Egr-1 as a transcriptional activator of the Ppp1r1b gene and provides insight into the molecular mechanisms that regulate medium spiny neuron maturation.

Neurotrophins and modifiers of chromatin acetylation and deacetylation participate in regulation of transcription during neuronal maturation and maintenance. The striatal medium spiny neuron is supported by cortically-derived brain derived neurotrophic factor and is the most vulnerable neuron in Huntington’s disease, in which growth factor and histone deacetylase activity are both disrupted. We examined the ability of three histone deacetylase inhibitors, trichostatin A, valproic acid and Compound 4 b, alone and combined with brain derived neurotrophic factor (BDNF), to promote phenotypic maturation of striatal medium spiny neurons in vitro. Exposure of these neurons to each of the three compounds led to an increase in overall histone H3 and H4 acetylation, dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa (DARPP-32) mRNA and protein, and mRNA levels of other markers of medium spiny neuron maturation. We were, however, unable to prove that HDAC inhibitors directly lead to remodeling of Ppp1r1b chromatin. In addition, induction of DARPP-32 by brain-derived neurotrophic factor was inhibited by histone deacetylase inhibitors. Although BDNF-induced increases in pTrkB, pAkt, pERK and Egr-1 were unchanged by combined application with VPA, the increase in DARPP-32 was relatively diminished. Strikingly, the NGF1A-binding protein, Nab2, was induced by BDNF, but not in the presence of VPA or TSA. Gel shift analysis showed that α-Nab2 super-shifted a band that is more prominent with extract derived from BDNF-treated neurons than with extracts from cultures treated with VPA alone or VPA plus BDNF. In addition, overexpression of Nab2 induced DARPP-32. We conclude that histone deacetylase inhibitors inhibit the induction of Nab2 by BDNF, and thereby the relative induction of DARPP-32.

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