Proteasome phosphorylation regulates cocaine-induced sensitization.

Repeated exposure to cocaine produces structural and functional modifications at synapses from neurons in several brain regions including the nucleus accumbens. These changes are thought to underlie cocaine-induced sensitization. The ubiquitin proteasome system plays a crucial role in the remodeling of synapses and has recently been implicated in addiction-related behavior. The ATPase Rpt6 subunit of the 26S proteasome is phosphorylated by Ca2+/calmodulin-dependent protein kinases II alpha at ser120 which is thought to regulate proteasome activity and distribution in neurons. Here, we demonstrate that Rpt6 phosphorylation is involved in cocaine-induced locomotor sensitization. Cocaine concomitantly increases proteasome activity and Rpt6 S120 phosphorylation in cultured neurons and in various brain regions of wild type mice including the nucleus accumbens and prefrontal cortex. In contrast, cocaine does not increase proteasome activity in Rpt6 phospho-mimetic (ser120Asp) mice. Strikingly, we found a complete absence of cocaine-induced locomotor sensitization in the Rpt6 ser120Asp mice. Together, these findings suggest a critical role for Rpt6 phosphorylation and proteasome function in the regulation cocaine-induced behavioral plasticity

Neurodevelopmental Changes in Excitatory Synaptic Structure and Function in the Cerebral Cortex of Sanfilippo Syndrome IIIA Mice.

Sanfilippo syndrome, MPS IIIA-D, results from deficits in lysosomal enzymes that specifically degrade heparan sulfate, a sulfated glycosaminoglycan. The accumulation of heparan sulfate results in neurological symptoms, culminating in extensive neurodegeneration and early death. To study the impact of storage in postnatal neurodevelopment, we examined murine models of MPS IIIA, which lack the enzyme sulfamidase. We show that changes occur in excitatory postsynaptic structure and function in the somatosensory cortex prior to signs of neurodegeneration. These changes coincide with accumulation of heparan sulfate with characteristic non-reducing ends, which is present at birth in the mutant mice. Accumulation of heparan sulfate was also detected in primary cultures of cortical neural cells, especially astrocytes. Accumulation of heparan sulfate in cultured astrocytes corresponded with augmented extracellular heparan sulfate and glypican 4 levels. Heparan sulfate from the cerebral cortex of MPS IIIA mice showed enhanced ability to increase glutamate AMPA receptor subunits at the cell surface of wild type neurons. These data support the idea that abnormalities in heparan sulfate content and distribution contribute to alterations in postsynaptic function. Our findings identify a disease-induced developmental phenotype that temporally overlaps with the onset of behavioral changes in a mouse model of MPS IIIA

Neurodevelopmental Changes in Excitatory Synaptic Structure and Function in the Cerebral Cortex of Sanfilippo Syndrome IIIA Mice.

Sanfilippo syndrome, MPS IIIA-D, results from deficits in lysosomal enzymes that specifically degrade heparan sulfate, a sulfated glycosaminoglycan. The accumulation of heparan sulfate results in neurological symptoms, culminating in extensive neurodegeneration and early death. To study the impact of storage in postnatal neurodevelopment, we examined murine models of MPS IIIA, which lack the enzyme sulfamidase. We show that changes occur in excitatory postsynaptic structure and function in the somatosensory cortex prior to signs of neurodegeneration. These changes coincide with accumulation of heparan sulfate with characteristic non-reducing ends, which is present at birth in the mutant mice. Accumulation of heparan sulfate was also detected in primary cultures of cortical neural cells, especially astrocytes. Accumulation of heparan sulfate in cultured astrocytes corresponded with augmented extracellular heparan sulfate and glypican 4 levels. Heparan sulfate from the cerebral cortex of MPS IIIA mice showed enhanced ability to increase glutamate AMPA receptor subunits at the cell surface of wild type neurons. These data support the idea that abnormalities in heparan sulfate content and distribution contribute to alterations in postsynaptic function. Our findings identify a disease-induced developmental phenotype that temporally overlaps with the onset of behavioral changes in a mouse model of MPS IIIA

Neurodevelopmental Changes in Excitatory Synaptic Structure and Function in the Cerebral Cortex of Sanfilippo Syndrome IIIA Mice

Sanfilippo syndrome, MPS IIIA-D, results from deficits in lysosomal enzymes that specifically degrade heparan sulfate, a sulfated glycosaminoglycan. The accumulation of heparan sulfate results in neurological symptoms, culminating in extensive neurodegeneration and early death. To study the impact of storage in postnatal neurodevelopment, we examined murine models of MPS IIIA, which lack the enzyme sulfamidase. We show that changes occur in excitatory postsynaptic structure and function in the somatosensory cortex prior to signs of neurodegeneration. These changes coincide with accumulation of heparan sulfate with characteristic non-reducing ends, which is present at birth in the mutant mice. Accumulation of heparan sulfate was also detected in primary cultures of cortical neural cells, especially astrocytes. Accumulation of heparan sulfate in cultured astrocytes corresponded with augmented extracellular heparan sulfate and glypican 4 levels. Heparan sulfate from the cerebral cortex of MPS IIIA mice showed enhanced ability to increase glutamate AMPA receptor subunits at the cell surface of wild type neurons. These data support the idea that abnormalities in heparan sulfate content and distribution contribute to alterations in postsynaptic function. Our findings identify a disease-induced developmental phenotype that temporally overlaps with the onset of behavioral changes in a mouse model of MPS IIIA

Altered Phosphorylation of the Proteasome Subunit Rpt6 Has Minimal Impact on Synaptic Plasticity and Learning.

Dynamic control of protein degradation via the ubiquitin proteasome system (UPS) is thought to play a crucial role in neuronal function and synaptic plasticity. The proteasome subunit Rpt6, an AAA ATPase subunit of the 19S regulatory particle (RP), has emerged as an important site for regulation of 26S proteasome function in neurons. Phosphorylation of Rpt6 on serine 120 (S120) can stimulate the catalytic rate of substrate degradation by the 26S proteasome and this site is targeted by the plasticity-related kinase Ca2+/calmodulin-dependent kinase II (CaMKII), making it an attractive candidate for regulation of proteasome function in neurons. Several in vitro studies have shown that altered Rpt6 S120 phosphorylation can affect the structure and function of synapses. To evaluate the importance of Rpt6 S120 phosphorylation in vivo, we created two mouse models which feature mutations at S120 that block or mimic phosphorylation at this site. We find that peptidase and ATPase activities are upregulated in the phospho-mimetic mutant and downregulated in the phospho-dead mutant [S120 mutated to aspartic acid (S120D) or alanine (S120A), respectively]. Surprisingly, these mutations had no effect on basal synaptic transmission, long-term potentiation (LTP), and dendritic spine dynamics and density in the hippocampus. Furthermore, these mutants displayed no deficits in cued and contextual fear memory. Thus, in a mouse model that blocks or mimics phosphorylation at this site, either compensatory mechanisms negate these effects, or small variations in proteasome activity are not enough to induce significant changes in synaptic structure, plasticity, or behavior

Altered Phosphorylation of the Proteasome Subunit Rpt6 Has Minimal Impact on Synaptic Plasticity and Learning.

Dynamic control of protein degradation via the ubiquitin proteasome system (UPS) is thought to play a crucial role in neuronal function and synaptic plasticity. The proteasome subunit Rpt6, an AAA ATPase subunit of the 19S regulatory particle (RP), has emerged as an important site for regulation of 26S proteasome function in neurons. Phosphorylation of Rpt6 on serine 120 (S120) can stimulate the catalytic rate of substrate degradation by the 26S proteasome and this site is targeted by the plasticity-related kinase Ca2+/calmodulin-dependent kinase II (CaMKII), making it an attractive candidate for regulation of proteasome function in neurons. Several in vitro studies have shown that altered Rpt6 S120 phosphorylation can affect the structure and function of synapses. To evaluate the importance of Rpt6 S120 phosphorylation in vivo, we created two mouse models which feature mutations at S120 that block or mimic phosphorylation at this site. We find that peptidase and ATPase activities are upregulated in the phospho-mimetic mutant and downregulated in the phospho-dead mutant [S120 mutated to aspartic acid (S120D) or alanine (S120A), respectively]. Surprisingly, these mutations had no effect on basal synaptic transmission, long-term potentiation (LTP), and dendritic spine dynamics and density in the hippocampus. Furthermore, these mutants displayed no deficits in cued and contextual fear memory. Thus, in a mouse model that blocks or mimics phosphorylation at this site, either compensatory mechanisms negate these effects, or small variations in proteasome activity are not enough to induce significant changes in synaptic structure, plasticity, or behavior

Enhanced activity of Alzheimer disease-associated variant of protein kinase Cα drives cognitive decline in a mouse model

Exquisitely tuned activity of protein kinase C (PKC) isozymes is essential to maintaining cellular homeostasis. Whereas loss-of-function mutations are generally associated with cancer, gain-of-function variants in one isozyme, PKCα, are associated with Alzheimer's disease (AD). Here we show that the enhanced activity of one variant, PKCα M489V, is sufficient to rewire the brain phosphoproteome, drive synaptic degeneration, and impair cognition in a mouse model. This variant causes a modest 30% increase in catalytic activity without altering on/off activation dynamics or stability, underscoring that enhanced catalytic activity is sufficient to drive the biochemical, cellular, and ultimately cognitive effects observed. Analysis of hippocampal neurons from PKCα M489V mice reveals enhanced amyloid-β-induced synaptic depression and reduced spine density compared to wild-type mice. Behavioral studies reveal that this mutation alone is sufficient to impair cognition, and, when coupled to a mouse model of AD, further accelerates cognitive decline. The druggability of protein kinases positions PKCα as a promising therapeutic target in AD

Guanidinylated Neomycin Conjugation Enhances Intranasal Enzyme Replacement in the Brain.

Iduronidase (IDUA)-deficient mice accumulate glycosaminoglycans in cells and tissues and exhibit many of the same neuropathological symptoms of patients suffering from Mucopolysaccharidosis I. Intravenous enzyme-replacement therapy for Mucopolysaccharidosis I ameliorates glycosaminoglycan storage and many of the somatic aspects of the disease but fails to treat neurological symptoms due to poor transport across the blood-brain barrier. In this study, we examined the delivery of IDUA conjugated to guanidinoneomycin (GNeo), a molecular transporter. GNeo-IDUA and IDUA injected intravenously resulted in reduced hepatic glycosaminoglycan accumulation but had no effect in the brain due to fast clearance from the circulation. In contrast, intranasally administered GNeo-IDUA entered the brain rapidly. Repetitive intranasal treatment with GNeo-IDUA reduced glycosaminoglycan storage, lysosome size and number, and neurodegenerative astrogliosis in the olfactory bulb and primary somatosensory cortex, whereas IDUA was less effective. The enhanced efficacy of GNeo-IDUA was not the result of increased nose-to-brain delivery or enzyme stability, but rather due to more efficient uptake into neurons and astrocytes. GNeo conjugation also enhanced glycosaminoglycan clearance by intranasally delivered sulfamidase to the brain of sulfamidase-deficient mice, a model of Mucopolysaccharidosis IIIA. These findings suggest the general utility of the guanidinoglycoside-based delivery system for restoring missing lysosomal enzymes in the brain

Guanidinylated Neomycin Conjugation Enhances Intranasal Enzyme Replacement in the Brain

Iduronidase (IDUA)-deficient mice accumulate glycosaminoglycans in cells and tissues and exhibit many of the same neuropathological symptoms of patients suffering from Mucopolysaccharidosis I. Intravenous enzyme-replacement therapy for Mucopolysaccharidosis I ameliorates glycosaminoglycan storage and many of the somatic aspects of the disease but fails to treat neurological symptoms due to poor transport across the blood-brain barrier. In this study, we examined the delivery of IDUA conjugated to guanidinoneomycin (GNeo), a molecular transporter. GNeo-IDUA and IDUA injected intravenously resulted in reduced hepatic glycosaminoglycan accumulation but had no effect in the brain due to fast clearance from the circulation. In contrast, intranasally administered GNeo-IDUA entered the brain rapidly. Repetitive intranasal treatment with GNeo-IDUA reduced glycosaminoglycan storage, lysosome size and number, and neurodegenerative astrogliosis in the olfactory bulb and primary somatosensory cortex, whereas IDUA was less effective. The enhanced efficacy of GNeo-IDUA was not the result of increased nose-to-brain delivery or enzyme stability, but rather due to more efficient uptake into neurons and astrocytes. GNeo conjugation also enhanced glycosaminoglycan clearance by intranasally delivered sulfamidase to the brain of sulfamidase-deficient mice, a model of Mucopolysaccharidosis IIIA. These findings suggest the general utility of the guanidinoglycoside-based delivery system for restoring missing lysosomal enzymes in the brain