Mechanisms and Consequences of Dopamine Depletion-Induced Attenuation of the Spinophilin/Neurofilament Medium Interaction.

Signaling changes that occur in the striatum following the loss of dopamine neurons in the Parkinson disease (PD) are poorly understood. While increases in the activity of kinases and decreases in the activity of phosphatases have been observed, the specific consequences of these changes are less well understood. Phosphatases, such as protein phosphatase 1 (PP1), are highly promiscuous and obtain substrate selectivity via targeting proteins. Spinophilin is the major PP1-targeting protein enriched in the postsynaptic density of striatal dendritic spines. Spinophilin association with PP1 is increased concurrent with decreases in PP1 activity in an animal model of PD. Using proteomic-based approaches, we observed dopamine depletion-induced decreases in spinophilin binding to multiple protein classes in the striatum. Specifically, there was a decrease in the association of spinophilin with neurofilament medium (NF-M) in dopamine-depleted striatum. Using a heterologous cell line, we determined that spinophilin binding to NF-M required overexpression of the catalytic subunit of protein kinase A and was decreased by cyclin-dependent protein kinase 5. Functionally, we demonstrate that spinophilin can decrease NF-M phosphorylation. Our data determine mechanisms that regulate, and putative consequences of, pathological changes in the association of spinophilin with NF-M that are observed in animal models of PD.

Loss of Thr286 phosphorylation disrupts synaptic CaMKIIα targeting, NMDAR activity and behavior in pre-adolescent mice.

In order to provide insight into in vivo roles of CaMKIIα autophosphorylation at Thr286 during postnatal development, behavioral, biochemical, and electrophysiological phenotypes of pre-adolescent Thr286 to Ala CaMKIIα knock-in (T286A-KI) and WT mice were examined. T286A-KI mice displayed cognitive deficits in a novel object recognition test and an anxiolytic phenotype in the elevated plus maze, suggesting disruption of normal developmental processes. At the molecular level, the ratio of total CaMKIIα to CaMKIIß in hippocampal lysates was significantly decreased≈2-fold in T286A-KI mice, and levels of both isoforms in synaptic subcellular fractions were decreased by≈80%. Total levels of GluA1 AMPA-glutamate receptor subunits and phosphorylation of GluA1 at the CaMKII site (Ser831) in synaptic fractions were unaltered, as were the frequency and amplitude of AMPAR-mediated spontaneous excitatory postsynaptic currents at hippocampal CA3-CA1 synapses. Synaptic levels of NMDA-glutamate receptor GluN1, GluN2A and GluN2B subunits also were unaltered. However, the reduced ratio of CaMKII to NMDAR subunits in synaptic fractions was linked to increased synaptic NMDAR-mediated currents in T286A-KI mice, apparently due to increased functional contributions by GluN2B NMDARs (assessed by Ro 25-6981 sensitivity). Thus, disruption of CaMKII synaptic targeting caused by elimination of Thr286 autophosphorylation leads to synaptic and behavioral deficits during pre-adolescence.

Age-dependent alterations in fibrillar collagen content and myocardial diastolic function: role of SPARC in post-synthetic procollagen processing.

Advanced age, independent of concurrent cardiovascular disease, can be associated with increased extracellular matrix (ECM) fibrillar collagen content and abnormal diastolic function. However, the mechanisms causing this left ventricular (LV) remodeling remain incompletely defined. We hypothesized that one determinant of age-dependent remodeling is a change in the extent to which newly synthesized procollagen is processed into mature collagen fibrils. We further hypothesized that secreted protein acidic and rich in cysteine (SPARC) plays a key role in the changes in post-synthetic procollagen processing that occur in the aged myocardium. Young (3 mo old) and old (18-24 mo old) wild-type (WT) and SPARC-null mice were studied. LV collagen content was measured histologically by collagen volume fraction, collagen composition was measured by hydroxyproline assay as soluble collagen (1 M NaCl extractable) versus insoluble collagen (mature cross-linked), and collagen morphological structure was examined by scanning electron microscopy. SPARC expression was measured by immunoblot analysis. LV and myocardial structure and function were assessed using echocardiographic and papillary muscle experiments. In WT mice, advanced age increased SPARC expression, myocardial diastolic stiffness, fibrillar collagen content, and insoluble collagen. In SPARC-null mice, advanced age also increased myocardial diastolic stiffness, fibrillar collagen content, and insoluble collagen but significantly less than those seen in WT old mice. As a result, insoluble collagen and myocardial diastolic stiffness were lower in old SPARC-null mice (1.36 +/- 0.08 mg hydroxyproline/g dry wt and 0.04 +/- 0.005) than in old WT mice (1.70 +/- 0.10 mg hydroxyproline/g dry wt and 0.07 +/- 0.005, P < 0.05). In conclusion, the absence of SPARC reduced age-dependent alterations in ECM fibrillar collagen and diastolic function. These data support the hypothesis that SPARC plays a key role in post-synthetic procollagen processing and contributes to the increase in collagen content found in the aged myocardium.

Pressure overload-induced alterations in fibrillar collagen content and myocardial diastolic function: role of secreted protein acidic and rich in cysteine (SPARC) in post-synthetic procollagen processing.

BACKGROUND: Chronic pressure overload causes myocardial hypertrophy, increased fibrillar collagen content, and abnormal diastolic function. We hypothesized that one determinant of these pressure overload-induced changes is the extracellular processing of newly synthesized procollagen into mature collagen fibrils. We further hypothesized that secreted protein acidic and rich in cysteine (SPARC) plays a key role in post-synthetic procollagen processing in normal and pressure-overloaded myocardium. METHODS AND RESULTS: To determine whether pressure overload-induced changes in collagen content and diastolic function are affected by the absence of SPARC, age-matched wild-type (WT) and SPARC-null mice underwent either transverse aortic constriction (TAC) for 4 weeks or served as nonoperated controls. Left ventricular (LV) collagen content was measured histologically by collagen volume fraction, collagen composition was measured by hydroxyproline assay as soluble collagen (1 mol/L NaCl extractable) versus insoluble collagen (mature cross-linked collagen), and collagen morphological structure was examined by scanning electron microscopy. SPARC expression was measured by immunoblot. LV, myocardial, and cardiomyocyte structure and function were assessed by echocardiographic, papillary muscle, and isolated cardiomyocyte studies. In WT mice, TAC increased LV mass, SPARC expression, myocardial diastolic stiffness, fibrillar collagen content, and soluble and insoluble collagen. In SPARC-null mice, TAC increased LV mass to an extent similar to WT mice. In addition, in SPARC-null mice, TAC increased fibrillar collagen content, albeit significantly less than that seen in WT TAC mice. Furthermore, the proportion of LV collagen that was insoluble was less in the SPARC-null TAC mice (86+/-2%) than in WT TAC mice (99+/-2%, P<0.05), and the proportion of collagen that was soluble was greater in the SPARC-null TAC mice (14+/-2%) than in WT TAC mice (1+/-2%, P<0.05) As a result, myocardial diastolic stiffness was lower in SPARC-null TAC mice (0.075+/-0.005) than in WT TAC mice (0.045+/-0.005, P<0.05). CONCLUSIONS: The absence of SPARC reduced pressure overload-induced alterations in extracellular matrix fibrillar collagen and diastolic function. These data support the hypothesis that SPARC plays a key role in post-synthetic procollagen processing and the development of mature cross-linked collagen fibrils in normal and pressure-overloaded myocardium.

SPARC regulates processing of procollagen I and collagen fibrillogenesis in dermal fibroblasts.

A characterization of the factors that control collagen fibril formation is critical for an understanding of tissue organization and the mechanisms that lead to fibrosis. SPARC (secreted protein acidic and rich in cysteine) is a counter-adhesive protein that binds collagens. Herein we show that collagen fibrils in SPARC-null skin from mice 1 month of age were inefficient in fibril aggregation and accumulated in the diameter range of 60-70 nm, a proposed intermediate in collagen fibril growth. In vitro, procollagen I produced by SPARC-null dermal fibroblasts demonstrated an initial preferential association with cell layers, in comparison to that produced by wild-type fibroblasts. However, the collagen I produced by SPARC-null cells was not efficiently incorporated into detergent-insoluble fractions. Coincident with an initial increase in cell association, greater amounts of total collagen I were present as processed forms in SPARC-null versus wild-type cells. Addition of recombinant SPARC reversed collagen I association with cell layers and decreased the processing of procollagen I in SPARC-null cells. Although collagen fibers formed on the surface of SPARC-null fibroblasts earlier than those on wild-type cells, fibers on SPARC-null fibroblasts did not persist. We conclude that SPARC mediates the association of procollagen I with cells, as well as its processing and incorporation into the extracellular matrix.