Analysis of the neuromuscular deficits caused by STAM1 deficiency.

The endosomal sorting complexes required for transport (ESCRT) pathway is composed of a series of protein complexes that are essential for sorting cargo through the endosome. In neurons, the ESCRT pathway is a key mediator of many cellular pathways that regulate neuronal morphogenesis as well as synaptic growth and function. The ESCRT-0 complex, consisting of HGS (hepatocyte growth factor-regulated tyrosine kinase substrate) and STAM (signal-transducing adaptor molecule), acts as a gate keeper to this pathway, ultimately determining the fate of the endosomal cargo. We previously showed that a single nucleotide substitution in Hgs results in structural and functional changes in the nervous system of teetering mice. To determine if these changes occurred as a function of HGS's role in the ESCRT pathway and its association with STAM1, we investigated if STAM1 deficiency also leads to a similar impairment of the nervous system. In contrast to teetering mice that die within 5 weeks of age and exhibit reduced body mass, 1-month-old Stam1 knockout mice were not visibly different from controls. However, by 3 months of age, STAM1 deficiency caused reduced muscle mass, strength, and motor performance. These changes in motor function did not correlate with either a loss in motor neuron number or abnormal myelination of peripheral nerves. Instead, the motor endplate structure was altered in the Stam1 knockout mice by 1 month of age and continued to degenerate over time, correlating with a significant reduction in muscle fiber size and increased expression of the embryonic γ acetylcholine receptor (AChR) subunit at 3 months of age. There was also a significant reduction in the levels of two presynaptic SNARE proteins, VTI1A and VAMP2, in the motor neurons of the Stam1 knockout mice. As loss of STAM1 expression replicates many of the structural changes at the motor endplates that we have previously reported with loss of HGS, these results suggest that the HGS/STAM1 complex plays a critical role in maintaining synaptic structure and function in the mammalian nervous system.

Excitatory and inhibitory imbalances in the trisynaptic pathway in the hippocampus in schizophrenia: a postmortem ultrastructural study.

BACKGROUND: A preponderance of evidence suggests that the hippocampus is a key region of dysfunction in schizophrenia. Neuroimaging and other studies indicate a relationship between hippocampal dysfunction and the degree of psychosis. Clinical data indicate hyperactivity in the hippocampus that precedes the onset of psychosis, and is correlated with symptom severity. In this study, we sought to identify circuitry at the electron microscopic level that could contribute to region-specific imbalances in excitation and inhibition in the hippocampus in schizophrenia. We used postmortem tissue from the anterior hippocampus from patients with schizophrenia and matched controls. Using stereological techniques, we counted and measured synapses, postsynaptic densities (PSDs), and evaluated size, number and optical density of mitochondria and parvalbumin-containing interneurons in key nodes of the trisynaptic pathway. Compared to controls, the schizophrenia group had decreased numbers of inhibitory synapses in CA3 and increased numbers of excitatory synapses in CA1; together, this indicates deficits in inhibition and an increase in excitation. The thickness of the PSD was larger in excitatory synapses in CA1, suggesting greater synaptic strength. In the schizophrenia group, there were fewer mitochondria in the dentate gyrus and a decrease in the optical density, a measure of functional integrity, in CA1. The number and optical density of parvalbumin interneurons were lower in CA3. The results suggest region-specific increases in excitatory circuitry, decreases in inhibitory neurotransmission and fewer or damaged mitochondria. These results are consistent with the hyperactivity observed in the hippocampus in schizophrenia in previous studies.

Evidence for upregulation of excitatory synaptic transmission in the substantia nigra in Schizophrenia: a postmortem ultrastructural study.

The dopamine hypothesis of schizophrenia suggests that psychotic symptoms originate from dysregulation of dopaminergic activity, which may be controlled by upstream innervation. We hypothesized that we would find anatomical evidence for the hyperexcitability seen in the SN. We examined and quantified synaptic morphology, which correlates with function, in the postmortem substantia nigra (SN) from 15 schizophrenia and 12 normal subjects. Synapses were counted using stereological techniques and classified based on the morphology of the post-synaptic density (PSD) and the presence or absence of a presynaptic density. The density and proportion of excitatory synapses was higher in the schizophrenia group than in controls, while the proportion (but not density) of inhibitory synapses was lower. We also detected in the schizophrenia group an increase in density of synapses with a PSD of intermediate thickness, which may represent excitatory synapses. The density of synapses with presynaptic densities was similar in both groups. The density of synapses with mixed morphologies was higher in the schizophrenia group than in controls. The human SN contains atypical synaptic morphology. We found an excess amount and proportion of excitatory synapses in the SN in schizophrenia that could result in hyperactivity and drive the psychotic symptoms of schizophrenia. The sources of afferent excitatory inputs to the SN arise from the subthalamic nucleus, the pedunculopontine nucleus, and the ventral tegmental area (VTA), areas that could be the source of excess excitation. Synapses with mixed morphologies may represent inputs from the VTA, which release multiple transmitters.

Increased glutamate transmission onto dorsal striatum spiny projection neurons in Pink1 knockout rats.

Loss-of-function PTEN Induced Kinase 1 (PINK1) mutations cause early-onset familial Parkinson's disease (PD) with similar clinical and neuropathological characteristics as idiopathic PD. While Pink1 knockout (KO) rats have mitochondrial dysfunction, locomotor deficits, and α-synuclein aggregates in several brain regions such as cerebral cortex, dorsal striatum, and substantia nigra, the functional ramifications on synaptic circuits are unknown. Using whole cell patch clamp recordings, we found a significant increase in the frequency of spontaneous excitatory postsynaptic currents (sEPSCs) onto striatal spiny projection neurons (SPNs) in Pink1 KO rats at ages 4 and 6 months compared to wild-type (WT) littermates, suggesting increased excitability of presynaptic neurons. While sEPSC amplitudes were also increased at 2 and 4 months, no changes were observed in AMPAR/NMDAR ratio or receptor expression. Further analysis revealed increased glutamate release probability and decreased recovery of the synaptic vesicle pool following a train of stimulation in Pink1 KO rats. Ultrastructural analysis revealed increased excitatory and inhibitory synapse number and increased levels of presynaptic α-synuclein, while the number and structure of striatal mitochondria appeared normal. Lastly, we found that Pink1 KO rats have altered striatal dopamine tone, which together with the abnormal α- synuclein distribution and dysfunctional mitochondria, could contribute to the increase in excitatory transmission. Together, these studies show that PINK1 is necessary for normal glutamatergic transmission onto striatal SPNs and reveal possible mechanisms underlying striatal circuit dysfunction in PD.

Abnormalities in the copper transporter CTR1 in postmortem hippocampus in schizophrenia: A subregion and laminar analysis.

Dysbindin-1 modulates copper transport, which is crucial for cellular homeostasis. Several brain regions implicated in schizophrenia exhibit decreased levels of dysbindin-1, which may affect copper homeostasis therein. Our recent study showed decreased levels of dysbindin-1, the copper transporter-1 (CTR1) and copper in the substantia nigra in schizophrenia, providing the first evidence of disrupted copper transport in schizophrenia. In the present study, we hypothesized that there would be lower levels of dysbindin-1 and CTR1 in the hippocampus in schizophrenia versus a comparison group. Using semi-quantitative immunohistochemistry for dysbindin1 and CTR1, we measured the optical density in a layer specific fashion in the hippocampus and entorhinal cortex in ten subjects with schizophrenia and ten comparison subjects. Both regions were richly immunolabeled for CTR1 and dysbindin1 in both groups. In the superficial layers of the entorhinal cortex, CTR1 immunolabeled neuropil and cells showed lower optical density values in patients versus the comparison group. In the molecular layer of the dentate gyrus, patients had higher optical density values of CTR1 versus the comparison group. The density and distribution of dysbindin-1 immunolabeling was similar between groups. These laminar specific alterations of CTR1 in schizophrenia suggest abnormal copper transport in those locations.

Interactions between knockout of schizophrenia risk factor Dysbindin-1 and copper metabolism in mice.

BACKGROUND AND PURPOSE: DTNBP1 gene variation and lower dysbindin-1 protein are associated with schizophrenia. Previous evidence suggests that downregulated dysbindin-1 expression results in lower expression of copper transporters ATP7A (intracellular copper transporter) and SLC31A1 (CTR1; extracellular copper transporter), which are required for copper transport across the blood brain barrier. However, whether antipsychotic medications used for schizophrenia treatment may modulate these systems is unclear. EXPERIMENTAL APPROACH: The current study measured behavioral indices of neurological function in dysbindin-1 functional knockout (KO) mice and their wild-type (WT) littermates with or without quetiapine treatment. We assessed serum and brain copper levels, ATP7A and CTR1 mRNA, and copper transporter-expressing cellular population transcripts: TTR (transthyretin; choroid plexus epithelial cells), MBP (myelin basic protein; oligodendrocytes), and GJA1 (gap-junction protein alpha-1; astrocytes) in cortex and hippocampus. KEY RESULTS: Regardless of genotype, quetiapine significantly reduced TTR, MBP, CTR1 mRNA, and serum copper levels. Neurological function of untreated KO mice was abnormal, and ledge instability was rescued with quetiapine. KO mice were hyperactive after 10 min in the open-field assay, which was not affected by treatment. CONCLUSIONS AND IMPLICATIONS: Dysbindin-1 KO results in hyperactivity, altered serum copper, and neurological impairment, the last of which is selectively rescued with quetiapine. Antipsychotic treatment modulates specific cellular populations, affecting myelin, the choroid plexus, and copper transport across the blood brain barrier. Together these results indicate the widespread impact of antipsychotic treatment, and that alteration of dysbindin-1 may be sufficient, but not necessary, for specific schizophrenia pathology.

Evidence for altered excitatory and inhibitory tone in the post-mortem substantia nigra in schizophrenia.

Objectives: The substantia nigra (SN) receives glutamatergic and GABAergic inputs that regulate dopaminergic neuronal activity. Imaging studies have shown hyperactivity of the SN in schizophrenia (SZ) patients. We examined neurochemically defined inputs to the SN, synaptic density, and neuromelanin content that might contribute to or reflect this hyperexcitability.Methods: Glutamatergic axon terminals were identified by the immunohistochemical localisation of vGLUT1 and vGLUT2; GABA inputs were identified by the immunohistochemical localisation of GAD67. Neuromelanin granules are visible in unstained sections and thus were assessed in unstained sections. Optical densitometry was measured to assess the density of vGLUT1, vGLUT2 or GAD67 immunolabelled axon terminals and neuromelanin granules. Electron microscopy was used to quantify synaptic and mitochondrial density.Results: Compared to controls, SZ subjects had nonsignificant trends toward a decrease in vGLUT1, and an increase in both vGLUT2 and GAD67. vGLUT1 was negatively correlated with GAD67 in normal controls (NCs) and positively correlated in SZ subjects. A correlation of coefficient analysis showed a significant difference between the negative correlation in NCs and the positive correlation in SZ subjects. Frequency histograms showed the distribution of neuromelanin density was different in SZ subjects compared to NCs. Synaptic density data showed a decrease in inhibitory synapses in SZ subjects. Mitochondrial density was normal in SZ subjects.Conclusions: Synaptic density alterations and the lack of a positive correlation between GAD67 and vGLUT1 could contribute to hyperactivity in the SN.

Pathology of white matter integrity in three major white matter fasciculi: A post-mortem study of schizophrenia and treatment status.

BACKGROUND AND PURPOSE: Imaging studies have shown that people with schizophrenia exhibit abnormal connectivity termed "dysconnectivity" in several white matter tracts, including the cingulum bundle (CB), corpus callosum (CC), and arcuate fasciculus (AF). This study aimed to elucidate potential contributors to schizophrenia "dysconnectivity." EXPERIMENTAL APPROACH: Western blot analysis was used to compare protein levels of myelin basic protein, neurofilament heavy, autophagosome marker LC3, and microtubule marker α-tubulin in post-mortem human CB, CC, and AF in schizophrenia subjects (SZ) and matched normal controls (NC). Additionally, SZ cases were subdivided by treatment status: off-medication (OFF) or on-medication (ON). KEY RESULTS: In the CC, the combined SZ group exhibited less neurofilament heavy protein than the NCs. In the CB, the combined SZ group had similar levels of α-tubulin protein versus NC, but OFF subjects had increased α-tubulin protein versus ON and NCs. There were significant correlations between α-tubulin and all other proteins but only in the CB. The strong negative relationship between α-tubulin versus myelin basic protein and α-tubulin versus LC3 in NCs was absent in SZs; coefficients comparison showed significant differences. Preliminary race analyses revealed that African American SZ had less AF α-tubulin than Caucasian SZ and African American normal controls. CONCLUSIONS AND IMPLICATIONS: The results show a relationship between tract- and protein-specific abnormalities and diagnosis, treatment, and race. These data suggest there is a dysregulation of the relationship between α-tubulin and the other markers of white matter integrity observed in the CB in schizophrenia.