Native-state proteomics of Parvalbumin interneurons identifies unique molecular signatures and vulnerabilities to early Alzheimer's pathology.

Dysfunction in fast-spiking parvalbumin interneurons (PV-INs) may represent an early pathophysiological perturbation in Alzheimer's Disease (AD). Defining early proteomic alterations in PV-INs can provide key biological and translationally-relevant insights. We used cell-type-specific in-vivo biotinylation of proteins (CIBOP) coupled with mass spectrometry to obtain native-state PV-IN proteomes. PV-IN proteomic signatures include high metabolic and translational activity, with over-representation of AD-risk and cognitive resilience-related proteins. In bulk proteomes, PV-IN proteins were associated with cognitive decline in humans, and with progressive neuropathology in humans and the 5xFAD mouse model of Aß pathology. PV-IN CIBOP in early stages of Aß pathology revealed signatures of increased mitochondria and metabolism, synaptic and cytoskeletal disruption and decreased mTOR signaling, not apparent in whole-brain proteomes. Furthermore, we demonstrated pre-synaptic defects in PV-to-excitatory neurotransmission, validating our proteomic findings. Overall, in this study we present native-state proteomes of PV-INs, revealing molecular insights into their unique roles in cognitive resiliency and AD pathogenesis.

Exploratory mass spectrometry of cerebrospinal fluid from persons with autopsy-confirmed LATE-NC.

Background: Common neuropathologies associated with dementia include Alzheimer's disease neuropathologic change (ADNC) and limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC). Biofluid proteomics provides a window into the pathobiology of dementia and the information from biofluid tests may help guide clinical management. Methods: Participants were recruited from a longitudinal cohort of older adults at the University of Kentucky AD Research Center. A convenience sample of clinically obtained lumbar puncture cerebrospinal fluid (CSF) samples was analyzed from 29 older adults that had autopsy confirmation of the presence or absence of LATE-NC. Nine of the participants had autopsy-confirmed LATE-NC. Antemortem CSF specimens were analyzed in two separate processes: From one group, aliquots were depleted of highly abundant proteins using affinity spin columns. Tryptic digests of sample proteins were subjected to liquid chromatographic separation and mass spectrometry using an Eksigent Ekspert nanoLC 400 system in line with a Sciex 6600+ mass spectrometer. Protein identification was performed using Protein Pilot (Sciex, ver. 5) software, and relative quantification was performed using the SWATH processing microApp in PeakView and MarkerView software (Sciex), respectively. Following data analyses, additional studies were performed using western blots. Results: A total of 830 proteins were identified in the samples depleted of abundant proteins, and 730 proteins were identified in the non-depleted samples. Whereas some dementia-related proteins were detected (Aß peptide and α-synuclein protein), others were not (TDP-43, TMEM106B, and tau proteins). When the Bonferroni correction was applied to correct for multiple comparisons, only 4 proteins showed differential expression (LATE-NC vs non-LATE-NC) in the nondepleted samples (RBP4, MIF, IGHG3 and ITM2B), whereas none showed statistically different changes in the depleted samples. Post-hoc western blots confirmed that RBP4 expression was higher in the LATE-NC cases at the group level, but there was overlap between the levels of RBP4 in LATE-NC and non-LATE-NC cases. Conclusions: An exploratory assessment of CSF proteomes of autopsy-confirmed LATE-NC and non-LATE-NC cases from a community-based cohort failed to demonstrate a clear-cut proteomic fingerprint that distinguished the two groups. There was intriguing increase in RBP4 protein levels in CSF from LATE-NC cases. This may provide clues about pathogenetic mechanisms in LATE-NC.

A neuropathologic feature of brain aging: multi-lumen vascular profiles.

Cerebrovascular pathologies other than frank infarctions are commonly seen in aged brains. Here, we focus on multi-lumen vascular profiles (MVPs), which are characterized by multiple vessel lumens enclosed in a single vascular channel. Little information exists on the prevalence, risk factors, and co-pathologies of MVPs. Therefore, we used samples and data from the University of Kentucky Alzheimer's Disease Research Center (n = 91), the University of Kentucky Pathology Department (n = 31), and the University of Pittsburgh Pathology Department (n = 4) to study MVPs. Age at death was correlated with MVP density in the frontal neocortex, Brodmann Area 9 (r = 0.51; p < 0.0001). Exploratory analyses were performed to evaluate the association between conventional vascular risk factors (e.g., hypertension, diabetes), cardiovascular diseases (e.g., heart attack, arrhythmia), and cerebrovascular disease (e.g., stroke); the only nominal association with MVP density was a self-reported history of brain trauma (Prevalence Ratio = 2.1; 95 CI 1.1-3.9, before correcting for multiple comparisons). No specific associations were detected between neuropathological (e.g., brain arteriolosclerosis) or genetic (e.g., APOE) variables and MVP density. Using a tissue clearing method called SeeDB, we provide 3-dimensional images of MVPs in brain tissue. We conclude that MVPs are an age-related brain pathology and more work is required to identify their clinical-pathological correlation and associated risk factors.

Native-state proteomics of Parvalbumin interneurons identifies novel molecular signatures and metabolic vulnerabilities to early Alzheimer's disease pathology.

One of the earliest pathophysiological perturbations in Alzheimer's Disease (AD) may arise from dysfunction of fast-spiking parvalbumin (PV) interneurons (PV-INs). Defining early protein-level (proteomic) alterations in PV-INs can provide key biological and translationally relevant insights. Here, we use cell-type-specific in vivo biotinylation of proteins (CIBOP) coupled with mass spectrometry to obtain native-state proteomes of PV interneurons. PV-INs exhibited proteomic signatures of high metabolic, mitochondrial, and translational activity, with over-representation of causally linked AD genetic risk factors. Analyses of bulk brain proteomes indicated strong correlations between PV-IN proteins with cognitive decline in humans, and with progressive neuropathology in humans and mouse models of Aß pathology. Furthermore, PV-IN-specific proteomes revealed unique signatures of increased mitochondrial and metabolic proteins, but decreased synaptic and mTOR signaling proteins in response to early Aß pathology. PV-specific changes were not apparent in whole-brain proteomes. These findings showcase the first native state PV-IN proteomes in mammalian brain, revealing a molecular basis for their unique vulnerabilities in AD.

Neurodegenerative pathologies associated with behavioral and psychological symptoms of dementia in a community-based autopsy cohort.

In addition to the memory disorders and global cognitive impairment that accompany neurodegenerative diseases, behavioral and psychological symptoms of dementia (BPSD) commonly impair quality of life and complicate clinical management. To investigate clinical-pathological correlations of BPSD, we analyzed data from autopsied participants from the community-based University of Kentucky Alzheimer's Disease Research Center longitudinal cohort (n = 368 research volunteers met inclusion criteria, average age at death 85.4 years). Data assessing BPSD were obtained approximately annually, including parameters for agitation, anxiety, apathy, appetite problems, delusions, depression, disinhibition, hallucinations, motor disturbance, and irritability. Each BPSD was scored on a severity scale (0-3) via the Neuropsychiatric Inventory Questionnaire (NPI-Q). Further, Clinical Dementia Rating (CDR)-Global and -Language evaluations (also scored on 0-3 scales) were used to indicate the degree of global cognitive and language impairment. The NPI-Q and CDR ratings were correlated with neuropathology findings at autopsy: Alzheimer's disease neuropathological changes (ADNC), neocortical and amygdala-only Lewy bodies (LBs), limbic predominant age-related TDP-43 encephalopathy neuropathologic changes (LATE-NC), primary age-related tauopathy (PART), hippocampal sclerosis, and cerebrovascular pathologies. Combinations of pathologies included the quadruple misfolding proteinopathy (QMP) phenotype with co-occurring ADNC, neocortical LBs, and LATE-NC. Statistical models were used to estimate the associations between BPSD subtypes and pathologic patterns. Individuals with severe ADNC (particularly those with Braak NFT stage VI) had more BPSD, and the QMP phenotype was associated with the highest mean number of BPSD symptoms: > 8 different BPSD subtypes per individual. Disinhibition and language problems were common in persons with severe ADNC but were not specific to any pathology. "Pure" LATE-NC was associated with global cognitive impairment, apathy, and motor disturbance, but again, these were not specific associations. In summary, Braak NFT stage VI ADNC was strongly associated with BPSD, but no tested BPSD subtype was a robust indicator of any particular "pure" or mixed pathological combination.

Urinary Incontinence in a Community-Based Autopsy Cohort Is Associated with Limbic Predominant Age-Related TDP-43 Encephalopathy Neuropathologic Changes.

BACKGROUND: Dementia and urinary incontinence (UI) are etiologically complex clinical syndromes. Dementia and UI often occur in the same individuals, but underlying factors connecting them are incompletely understood. OBJECTIVE: Query data from a community-based autopsy series to assess pathologies that underlie UI. METHODS: Included research subjects came to autopsy from the University of Kentucky Alzheimer's Disease Research Center longitudinal cohort. A total of 368 research volunteers met inclusion criteria for this cross-sectional study. The average age at death was 85.3 years and the average number of annual clinic visits was 5.2 visits. Statistical models were run to evaluate which pathologies were associated with UI. Data included pathologies scored according to conventional stage-based systems, and these studies were complemented by quantitative digital neuropathology. RESULTS: Dementia was diagnosed at the final clinical visit in 208 (56.7% of the sample) and UI was documented in 156 (42.7%). UI was associated with depression and dementia (both p < 0.001). More women than men had a history of UI (p < 0.04), and women with UI had had more biological children than those without UI (p < 0.005). Participants with limbic predominant age-related TDP-43 encephalopathy neuropathologic changes (LATE-NC) were more likely to have UI than those without LATE-NC (p < 0.001). The presence of LATE-NC (Stage > 1) was associated with UI with or without severe Alzheimer's disease neuropathologic changes and/or Lewy body pathology. CONCLUSION: In this community-based autopsy cohort, multiple factors were associated with UI, but the neuropathologic change most robustly associated with UI was LATE-NC.

Brain Cell Type-Specific Nuclear Proteomics Is Imperative to Resolve Neurodegenerative Disease Mechanisms.

Neurodegenerative diseases (NDs) involve complex cellular mechanisms that are incompletely understood. Emerging findings have revealed that disruption of nuclear processes play key roles in ND pathogenesis. The nucleus is a nexus for gene regulation and cellular processes that together, may underlie pathomechanisms of NDs. Furthermore, many genetic risk factors for NDs encode proteins that are either present in the nucleus or are involved in nuclear processes (for example, RNA binding proteins, epigenetic regulators, or nuclear-cytoplasmic transport proteins). While recent advances in nuclear transcriptomics have been significant, studies of the nuclear proteome in brain have been relatively limited. We propose that a comprehensive analysis of nuclear proteomic alterations of various brain cell types in NDs may provide novel biological and therapeutic insights. This may be feasible because emerging technical advances allow isolation and investigation of intact nuclei from post-mortem frozen human brain tissue with cell type-specific and single-cell resolution. Accordingly, nuclei of various brain cell types harbor unique protein markers which can be used to isolate cell-type specific nuclei followed by down-stream proteomics by mass spectrometry. Here we review the literature providing a rationale for investigating proteomic changes occurring in nuclei in NDs and then highlight the potential for brain cell type-specific nuclear proteomics to enhance our understanding of distinct cellular mechanisms that drive ND pathogenesis.

Frequency of LATE neuropathologic change across the spectrum of Alzheimer's disease neuropathology: combined data from 13 community-based or population-based autopsy cohorts.

Limbic-predominant age-related TDP-43 encephalopathy neuropathologic change (LATE-NC) and Alzheimer's disease neuropathologic change (ADNC) are each associated with substantial cognitive impairment in aging populations. However, the prevalence of LATE-NC across the full range of ADNC remains uncertain. To address this knowledge gap, neuropathologic, genetic, and clinical data were compiled from 13 high-quality community- and population-based longitudinal studies. Participants were recruited from United States (8 cohorts, including one focusing on Japanese-American men), United Kingdom (2 cohorts), Brazil, Austria, and Finland. The total number of participants included was 6196, and the average age of death was 88.1 years. Not all data were available on each individual and there were differences between the cohorts in study designs and the amount of missing data. Among those with known cognitive status before death (n = 5665), 43.0% were cognitively normal, 14.9% had MCI, and 42.4% had dementia-broadly consistent with epidemiologic data in this age group. Approximately 99% of participants (n = 6125) had available CERAD neuritic amyloid plaque score data. In this subsample, 39.4% had autopsy-confirmed LATE-NC of any stage. Among brains with "frequent" neuritic amyloid plaques, 54.9% had comorbid LATE-NC, whereas in brains with no detected neuritic amyloid plaques, 27.0% had LATE-NC. Data on LATE-NC stages were available for 3803 participants, of which 25% had LATE-NC stage > 1 (associated with cognitive impairment). In the subset of individuals with Thal Aß phase = 0 (lacking detectable Aß plaques), the brains with LATE-NC had relatively more severe primary age-related tauopathy (PART). A total of 3267 participants had available clinical data relevant to frontotemporal dementia (FTD), and none were given the clinical diagnosis of definite FTD nor the pathological diagnosis of frontotemporal lobar degeneration with TDP-43 inclusions (FTLD-TDP). In the 10 cohorts with detailed neurocognitive assessments proximal to death, cognition tended to be worse with LATE-NC across the full spectrum of ADNC severity. This study provided a credible estimate of the current prevalence of LATE-NC in advanced age. LATE-NC was seen in almost 40% of participants and often, but not always, coexisted with Alzheimer's disease neuropathology.

Cell type-specific biotin labeling in vivo resolves regional neuronal and astrocyte proteomic differences in mouse brain.

Proteomic profiling of brain cell types using isolation-based strategies pose limitations in resolving cellular phenotypes representative of their native state. We describe a mouse line for cell type-specific expression of biotin ligase TurboID, for in vivo biotinylation of proteins. Using adenoviral and transgenic approaches to label neurons, we show robust protein biotinylation in neuronal soma and axons throughout the brain, allowing quantitation of over 2000 neuron-derived proteins spanning synaptic proteins, transporters, ion channels and disease-relevant druggable targets. Next, we contrast Camk2a-neuron and Aldh1l1-astrocyte proteomes and identify brain region-specific proteomic differences within both cell types, some of which might potentially underlie the selective vulnerability to neurological diseases. Leveraging the cellular specificity of proteomic labeling, we apply an antibody-based approach to uncover differences in neuron and astrocyte-derived signaling phospho-proteins and cytokines. This approach will facilitate the characterization of cell-type specific proteomes in a diverse number of tissues under both physiological and pathological states.

The Amygdala as a Locus of Pathologic Misfolding in Neurodegenerative Diseases.

Over the course of most common neurodegenerative diseases the amygdala accumulates pathologically misfolded proteins. Misfolding of 1 protein in aged brains often is accompanied by the misfolding of other proteins, suggesting synergistic mechanisms. The multiplicity of pathogenic processes in human amygdalae has potentially important implications for the pathogenesis of Alzheimer disease, Lewy body diseases, chronic traumatic encephalopathy, primary age-related tauopathy, and hippocampal sclerosis, and for the biomarkers used to diagnose those diseases. Converging data indicate that the amygdala may represent a preferential locus for a pivotal transition from a relatively benign clinical condition to a more aggressive disease wherein multiple protein species are misfolded. Thus, understanding of amygdalar pathobiology may yield insights relevant to diagnoses and therapies; it is, however, a complex and imperfectly defined brain region. Here, we review aspects of amygdalar anatomy, connectivity, vasculature, and pathologic involvement in neurodegenerative diseases with supporting data from the University of Kentucky Alzheimer's Disease Center autopsy cohort. Immunohistochemical staining of amygdalae for Aß, Tau, α-synuclein, and TDP-43 highlight the often-coexisting pathologies. We suggest that the amygdala may represent an "incubator" for misfolded proteins and that it is possible that misfolded amygdalar protein species are yet to be discovered.

Adenovirus 5 fibers mutated at the putative HSPG-binding site show restricted retargeting with targeting peptides in the HI loop.

Adenoviral vectors are commonly used for liver-directed gene therapy following systemic administration owing to their strong propensity for hepatocyte transduction. However, many disease applications would benefit from the delivery of adenoviruses to alternate tissues via this route. Research has thus focused on stripping the virus of native hepatic tropism in conjunction with modifying virus capsid proteins to incorporate novel tropism. Recently, the KO1S* adenovirus serotype 5 fiber mutant, devoid of both coxsackie and adenovirus receptor binding in the fiber knob domain and mutated at the putative heparan sulphate proteoglycan binding site in the fiber shaft, was shown to possess strikingly poor hepatic tropism in mice, rats, and non-human primates. Thus, it is an ideal candidate for retargeting strategies. We therefore assessed the ability of peptide-modified KO1S* fibers to retarget adenovirus. Peptide insertions were well tolerated and virions produced to high titers. However, expected retargeting at the level of transduction was not observed, despite cell-binding studies showing enhanced vector targeting at the cell surface. Cy3 labeling studies showed retarded trafficking of S*-containing fibers. Taken together, our data demonstrates that KO1S* mutant fibers are ineffective for cell retargeting strategies.

Judgment of disfluency in people who stutter and people who do not stutter: results from magnitude estimation.

Two experiments used a magnitude estimation paradigm to test whether perception of disfluency is a function of whether the speaker and the listener stutter or do not stutter. Utterances produced by people who stutter were judged as "less fluent," and, critically, this held for apparently fluent utterances as well as for utterances identified as containing disfluency. Additionally, people who stutter tended to perceive utterances as less fluent, independent of who produced these utterances. We argue that these findings are consistent with a view that articulatory differences between the speech of people who stutter and people who do not stutter lead to perceptually relevant vocal differences. We suggest that these differences are detected by the speech self-monitoring system (which uses speech perception) resulting in covert repairs. Our account therefore shares characteristics with the Covert Repair (Postma & Kolk, 1993) and Vicious Circle (Vasic & Wijnen, 2005) hypotheses. It differs from the Covert Repair hypothesis in that it no longer assumes an additional deficit at the phonological planning level. It differs from the Vicious Circle hypothesis in that it no longer attributes hypervigilant monitoring to unknown, external factors. Rather, the self-monitor becomes hypervigilant because the speaker is aware that his/her speech is habitually deviant, even when it is not, strictly speaking, disfluent.

Skewing of pretransplant anti-HLA class I antibodies of immunoglobulin G isotype solely toward immunoglobulin G1 subclass is associated with poorer renal allograft survival.

Sensitized patients with lymphocytotoxic immunoglobulin (Ig)G anti-human leukocyte antigen (HLA) antibodies have an increased risk of rejection and poorer graft survival. Little is known, however, about the correlation between IgG antibody subclass and clinical outcomes. We identified 20 sensitized renal transplant recipients (panel reactive antibody >15%), all of whom had anti-HLA class I antibodies of an IgG isotype with known specificity before transplantation but who received a crossmatch negative graft. We analyzed the degree of skewing solely toward IgG1 (n=11) or to other IgG subclasses with or without IgG1 (n=9) and correlated these findings with graft survival. At last follow-up (median follow-up 28 months), 6 of 11 patients (55%) with anti-HLA antibodies skewed toward IgG1 had lost their grafts compared with 0 of 9 patients (0%) with anti-HLA antibodies not skewed toward IgG1 (P =0.01 log-rank test). Anti-HLA antibodies of an IgG1 subclass may be a novel marker predicting poor graft outcome.

Will transplantation of kidneys from donors with blood group A2 into recipients with blood group B help British Indo-Asian patients with renal failure?

Despite a high incidence of renal failure, disproportionately fewer Indo-Asians in the United Kingdom receive a renal transplant, in part because of the high prevalence of blood group B. It is now clear that it is possible to safely transplant kidneys from donors with blood group A of the subtype A2 into recipients with blood group B if the latter have low titers of anti-A antibodies. We measured the anti-A titers in 25 Indo-Asian patients on dialysis being considered for transplantation and found stably low titers in all. Titers were comparable to those found in a control white population with blood group B. In addition, in a complement-dependent cytotoxicity crossmatch against group A lymphocytes, the only positive results were obtained in those with high preexisting panel reactivity (i.e., because of the presence of preformed anti-human leukocyte antigen antibodies). We conclude that there are grounds for investigating this approach further to solve the ethnic disparity in rates of transplantation.