BMP9 ameliorates amyloidosis and the cholinergic defect in a mouse model of Alzheimer’s disease Rebecca M. Burkea, Timothy A. Normana, Tarik F. Haydarb, Barbara E. Slacka, Susan E. Leemanc,1, Jan Krzysztof Blusztajna, and Tiffany J. Mellotta,1 aDepartment of Pathology and Laboratory Medicine, bDepartment of Anatomy and Neurobiology, and cDepartment of Pharmacology and Experimental Therapeutics, Boston University School of Medicine, Boston, MA 02118 Contributed by Susan E. Leeman, October 16, 2013 (sent for review June 21, 2013) Bone morphogenetic protein 9 (BMP9) promotes the acquisition of A prominent example of such an illness, for which effective the cholinergic phenotype in basal forebrain cholinergic neurons therapies are critically needed, is Alzheimer’s disease (AD). A (BFCN) during development and protects these neurons from decline in BFCN function and diminished cholinergic marker cholinergic dedifferentiation following axotomy when adminis- expression occurs in brains of patients with AD (6, 7) and is tered in vivo. A decline in BFCN function occurs in patients with similarly observed in AD animal models (6, 8–13). This dys- Alzheimer’s disease (AD) and contributes to the AD-associated function and/or degeneration of BFCN contribute to the mem- memory deficits. We infused BMP9 intracerebroventricularly for ory deficits that are a hallmark of AD (6, 7, 14). Some of the 7 d in transgenic AD model mice expressing green fluorescent pro- current treatments for AD are designed to increase the intra- tein specifically in cholinergic neurons (APP.PS1/CHGFP) and in synaptic levels of ACh by inhibiting the enzyme acetylcholines- wild-type littermate controls (WT/CHGFP). We used 5-mo-old mice, terase that breaks down the neurotransmitter (15). This approach, an age when the AD transgenics display early amyloid deposition however, is only mildly effective, presumably because the patho- and few cholinergic defects, and 10-mo-old mice, by which time physiologic process that underlies AD is progressive, leading to these mice exhibit established disease. BMP9 infusion reduced the inevitable loss of synapses (16, 17). Therefore, the use of trophic number of Aβ42-positive amyloid plaques in the hippocampus and factors [e.g., nerve growth factor (NGF)] (18) that support BFCN cerebral cortex of 5- and 10-mo-old APP.PS1/CHGFP mice and re- survival and maintain the differentiated state of these neurons versed the reductions in choline acetyltransferase protein levels in represents a possible strategy for the treatment of AD. the hippocampus of 10-mo-old APP.PS1/CHGFP mice. The treat- In the current study we explored the potential of BMP9 to ment increased cholinergic fiber density in the hippocampus of serve as a therapeutic agent for AD using the APPswe/PS1deltaE9 both WT/CHGFP and APP.PS1/CHGFP mice at both ages. BMP9 in- (APP.PS1) transgenic mouse model of AD. These mice were fusion also increased hippocampal levels of neurotrophin 3, insu- engineered to express murine amyloid precursor protein (APP) lin-like growth factor 1, and nerve growth factor and of the nerve with the human β-amyloid (Aβ) amino acid sequence harboring growth factor receptors, tyrosine kinase receptor A and p75/NGFR, mutations that cause a familial form of AD [the Swedish muta- irrespective of the genotype of the mice. These data show that tion APP(K595N/M596L): APPswe] together with a mutated BMP9 administration is effective in reducing the Aβ42 amyloid form of presenilin 1 (PS1 with exon 9 deleted: PS1dE9) (19). NEUROSCIENCE plaque burden, reversing cholinergic neuron abnormalities, and Although no model of AD fully recapitulates the human disease generating a neurotrophic milieu for BFCN in a mouse model of (20), APP.PS1 mice are well suited for our investigations because AD and provide evidence that the BMP9-signaling pathway may they exhibit (i) cholinergic defects (8–12), (ii) high production of constitute a therapeutic target for AD. Aβ peptides in brain and accumulation of amyloid plaques (21), and (iii) cognitive impairments (12, 22–26). We elected to study acetylcholine | APPswe PS1dE9 mice | growth/differentiation factor 2 | animals at 5 and 10 mo of age. At 5 mo, the APP.PS1 mice begin juvenile protective factors to exhibit amyloid accumulation (27) and have normal choline asal forebrain cholinergic neurons (BFCN) project to the Significance Bhippocampus and cerebral cortex where the release of their neurotransmitter, acetylcholine (ACh), is central for the pro- Bone morphogenetic protein 9 (BMP9) is a trophic factor for cesses of learning, memory, and attention throughout life (1). basal forebrain cholinergic neurons (BFCN) and constitutes a The acquisition of the cholinergic phenotype by these neurons candidate therapeutic molecule for diseases characterized by during development is induced by bone morphogenetic protein 9 BFCN dysfunction. A prominent example of such an illness, for (BMP9), also known as growth/differentiation factor 2 (GDF2), which effective therapies are critically needed, is Alzheimer’s which is expressed in the fetal basal forebrain (2). Application of disease (AD). A decline in BFCN function and diminished cholin- BMP9 to basal forebrain cell cultures and into the cerebral ergic marker expression occurs in brains of patients with AD and ventricles of developing mouse embryos (2) and adult mice (3) is observed in AD animal models. We report that BMP9 admin- significantly increases ACh production. Moreover, the idea that istration is effective in reducing the amyloid plaque burden, the BMP9-signaling pathway may be a master regulator of the reversing cholinergic neuron abnormalities, and generating a BFCN phenotype is supported by evidence showing that BMP9 is neurotrophic milieu for cholinergic neurons in a mouse model of sufficient to induce BFCN-like features in human embryonic AD, thus providing evidence that the BMP9-signaling pathway stem cell-derived neural progenitor cells (4) and that, in rodent may constitute a novel therapeutic target in AD. basal forebrain cell cultures, BMP9 induces the BFCN tran- scriptome (5). Recent studies indicate that intracerebroven- Author contributions: R.M.B., J.K.B., and T.J.M. designed research; R.M.B., T.A.N., T.F.H., B.E.S., J.K.B., and T.J.M. performed research; R.M.B., T.A.N., T.F.H., B.E.S., S.E.L., J.K.B., and T.J.M. tricularly (i.c.v.)-infused BMP9 fully prevents the cholinergic analyzed data; and R.M.B., B.E.S., S.E.L., J.K.B., and T.J.M. wrote the paper. dedifferentiation (i.e., loss of cholinergic markers) of axotomized The authors declare no conflict of interest. BFCN in mice (3). Taken together, these data provide a com- 1To whom correspondence may be addressed. E-mail: [email protected] or tmellott@bu. pelling motivation to determine the efficacy of BMP9 as a edu. treatment of diseases characterized by BFCN dysfunction and/ This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. or degeneration. 1073/pnas.1319297110/-/DCSupplemental. www.pnas.org/cgi/doi/10.1073/pnas.1319297110 PNAS | November 26, 2013 | vol. 110 | no. 48 | 19567–19572 Downloaded by guest on September 25, 2021 acetyltransferase (CHAT) activity, although they show some 5-Months : Anterior PBS PBS ) A ) 2 40 2 impairment of the BFCN projections (8). This time point rep- Male Female BMP9 25 BMP9 resents early stages of pathogenesis. The 10-mo-old APP.PS1 30 20 mice exhibit high amyloid deposition (27) and cholinergic im- PBS 15 pairment characterized by reduced CHAT activity and dystro- 20 phic cholinergic neurites in the terminal fields within the 10 10 42 Plaque Count/ROI (mm hippocampus and cerebral cortex (8). To facilitate studies of the 42 Plaque Count/ROI (mm 5 A BMP9 A cholinergic phenotype in this model, we crossed these mice with 0 0 Males Females Males Females a transgenic strain that expresses enhanced green fluorescent Somatosensory Cortex Anterior Hippocampus protein specifically in cholinergic cells (28, 29). Thus, our studies were performed either on wild-type control mice that did not B 5-Months: Posterior express the AD-related transgenes, designated WT/CHGFP, or Male Female on their AD model littermates, designated APP.PS1/CHGFP. Intracerebroventricular infusion of human recombinant BMP9 in these mice for a brief period of 7 d fully reversed AD-like PBS 14 PBS ) 30 ) 2 cholinergic pathology and induced the expression of procholi- PBS BMP9 2 BMP9 12 nergic growth factors including NGF and its receptors (p75/NGFR 25 10 20 and TRKA), neurotrophin 3 (NT3), and insulin-like growth factor 8 β 15 1 (IGF1). Unexpectedly, BMP9 infusion also reduced A 42 am- 6 10 yloid plaque number in the hippocampus and cerebral cortex of 4 42 Plaque Count/ROI (mm 42 Plaque Count/ROI (mm APP.PS1/CHGFP mice. Taken together, these data indicate that 5 2 A fi A BMP9 exhibits ef cacy as a therapeutic agent for AD. 0 0 BMP9 Males Females Males Females Primary Visual Cortex Posterior Hippocampus Results BMP9 Infusion Reduces the Number of Amyloid Plaques in the Hippo- campus and Cerebral Cortex. We determined the amyloid burden in APP.PS1/CHGFP mice using immunohistochemical staining. Fig. 1. BMP9 reduces the number of Aβ42 plaques in the hippocampus and Aβ42-positive plaques were present in the cortex and hippo- cortex of 5-mo-old APP.PS1/CHGFP mice. Immunohistochemical staining of Aβ42 campus of these animals. Consistent with previous studies in was performed on anterior (A)andposterior(B) brain sections from 5-mo-old APP.PS1 mice, the plaque number increased with age (30), and male and female APP.PS1/CHGFP mice as described in Materials and Methods. Representative images from each sex and treatment group are shown for the females were more vulnerable to the amyloidosis than the males anterior and posterior sections.