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Background: Phosphorylated cytoplasmic tau inclusions correlate with and precede cognitive deficits in Alzheimer’s disease (AD). However, pathological tau accumulation and relationships to synaptic changes remain unclear. Objective: To address this, we examined postmortem brain from 50 individuals with the full spectrum of AD (clinically and neuropathologically). Total tau, pTau231, and AMPA GluR1 were compared across two brain regions (entorhinal and middle frontal cortices), as well as clinically stratified groups (control, amnestic mild cognitive impairment, AD dementia), NIA-AA Alzheimer’s Disease Neuropathologic Change designations (Not, Low, Intermediate, High), and Braak tangle stages (1–6). Significant co-existing pathology was excluded to isolate changes attributed to pathologic AD. Methods: Synaptosomal fractionation and staining were performed to measure changes in total Tau, pTau231, and AMPA GluR1. Total Tau and pTau231 were quantified in synaptosomal fractions using Quanterix Simoa HD-X. Results: Increasing pTau231 in frontal postsynaptic fractions correlated positively with increasing clinical and neuropathological AD severity. Frontal cortex is representative of early AD, as it does not become involved by tau tangles until late in AD. Entorhinal total tau was significantly higher in the amnestic mild cognitive impairment group when compared to AD, but only after accounting for AD associated synaptic changes. Alterations in AMPA GluR1 observed in the entorhinal cortex, but not middle frontal cortex, suggest that pTau231 mislocalization and aggregation in postsynaptic structures may impair glutamatergic signaling by promoting AMPA receptor dephosphorylation and internalization. Conclusion: Results highlight the potential effectiveness of early pharmacological interventions targeting pTau231 accumulation at the postsynaptic density.

Concerns about the costs associated with autopsy assessment of Alzheimer disease and related dementias according to 2012 NIA-AA Guidelines have been expressed since the publication of those guidelines. For this reason, we designed and validated a Condensed Protocol for the neuropathologic diagnoses of Alzheimer disease neuropathologic change, Lewy Body disease neuropathologic change, as well as chronic microvascular lesions, hippocampal sclerosis of aging, and cerebral amyloid angiopathy. In this study, the Condensed Protocol is updated to include frontotemporal lobar degeneration [FTLD] tau (corticobasal degeneration, progressive supranuclear palsy, and Pick disease), FTLD-TDP, and limbic-predominant, age-related TDP-43 encephalopathy. The same 20 brain regions are sampled and processed in 5 tissue cassettes, which reduces reagent costs by approximately 65%. Three board-certified neuropathologists were blinded to the original Northwestern University Alzheimer's Disease Research Center Original Protocol neuropathological diagnoses and all clinical history information. The results yielded near uniform agreement with the original comprehensive Alzheimer's Disease Research Center neuropathologic assessments. Diagnostic sensitivity was not impacted. In summary, our recent results show that our updated Condensed Protocol is also an accurate and less expensive alternative to the comprehensive protocols for the additional neuropathologic diagnoses of FTLD Tau and TDP43 proteinopathies.

The TDP-43 type C pathological form of frontotemporal lobar degeneration is characterized by the presence of immunoreactive TDP-43 short and long dystrophic neurites, neuronal cytoplasmic inclusions, neuronal loss and gliosis and the absence of neuronal intranuclear inclusions. Frontotemporal lobar degeneration-TDP-type C cases are commonly associated with the semantic variant of primary progressive aphasia or behavioural variant frontotemporal dementia. Here, we provide detailed characterization of regional distributions of pathological TDP-43 and neuronal loss and gliosis in cortical and subcortical regions in 10 TDP-type C cases and investigate the relationship between inclusions and neuronal loss and gliosis. Specimens were obtained from the first 10 TDP-type C cases accessioned from the Northwestern Alzheimer’s Disease Research Center (semantic variant of primary progressive aphasia, n = 7; behavioural variant frontotemporal dementia, n = 3). A total of 42 cortical (majority bilateral) and subcortical regions were immunostained with a phosphorylated TDP-43 antibody and/or stained with haematoxylin–eosin. Regions were evaluated for atrophy, and for long dystrophic neurites, short dystrophic neurites, neuronal cytoplasmic inclusions, and neuronal loss and gliosis using a semiquantitative 5-point scale. We calculated a ‘neuron-to-inclusion’ score (TDP-type C mean score – neuronal loss and gliosis mean score) for each region per case to assess the relationship between TDP-type C inclusions and neuronal loss and gliosis. Primary progressive aphasia cases demonstrated leftward asymmetry of cortical atrophy consistent with the aphasic phenotype. We also observed abundant inclusions and neurodegeneration in both cortical and subcortical regions, with certain subcortical regions emerging as particularly vulnerable to dystrophic neurites (e.g. amygdala, caudate and putamen). Interestingly, linear mixed models showed that regions with lowest TDP-type C pathology had high neuronal dropout, and conversely, regions with abundant pathology displayed relatively preserved neuronal densities (P

Mitochondrial myopathies belong to a larger group of systemic diseases caused by morphological or biochemical abnormalities of mitochondria. Mitochondrial disorders can be caused by mutations in either the mitochondrial or nuclear genome. Only 5 % of all mitochondrial disorders are autosomal dominant. We analyzed DNA from members of the previously reported Puerto Rican kindred with an autosomal dominant mitochondrial myopathy (Heimann-Patterson et al. 1997). Linkage analysis suggested a putative locus on the pericentric region of the long arm of chromosome 22 (22q11). Using the tools of integrative genomics, we established chromosome 22 open reading frame 16 (C22orf16) (later designated as CHCHD10) as the only high-scoring mitochondrial candidate gene in our minimal candidate region. Sequence analysis revealed a double-missense mutation (R15S and G58R) in cis in CHCHD10 which encodes a coiled coil-helix-coiled coil-helix protein of unknown function. These two mutations completely co-segregated with the disease phenotype and were absent in 1,481 Caucasian and 80 Hispanic (including 32 Puerto Rican) controls. Expression profiling showed that CHCHD10 is enriched in skeletal muscle. Mitochondrial localization of the CHCHD10 protein was confirmed using immunofluorescence in cells expressing either wild-type or mutant CHCHD10. We found that the expression of the G58R, but not the R15S, mutation induced mitochondrial fragmentation. Our findings identify a novel gene causing mitochondrial myopathy, thereby expanding the spectrum of mitochondrial myopathies caused by nuclear genes. Our findings also suggest a role for CHCHD10 in the morphologic remodeling of the mitochondria.

A central feature of integrin interaction with physiologic ligands is the monodentate binding of a ligand carboxylate to a Mg2+ ion hexacoordinated at the metal ion-dependent adhesion site (MIDAS) in the integrin A domain. This interaction stabilizes the A domain in the high-affinity state, which is distinguished from the default low-affinity state by tertiary changes in the domain that culminate in cell adhesion. Small molecule ligand-mimetic integrin antagonists act as partial agonists, eliciting similar activating conformational changes in the A domain, which has contributed to paradoxical adhesion and increased patient mortality in large clinical trials. As with other ligand-mimetic integrin antagonists, the function-blocking mAb 107 binds MIDAS of integrin CD11b/CD18 A domain (CD11bA), but in contrast, it favors the inhibitory Ca2+ ion over the Mg2+ ion at MIDAS. We determined the crystal structures of the Fab fragment of mAb 107 complexed to the low- and high-affinity states of CD11bA. Favored binding of the Ca2+ ion at MIDAS is caused by the unusual symmetric bidentate ligation of a Fab-derived ligand Asp to a heptacoordinated MIDAS Ca2+ ion. Binding of the Fab fragment of mAb 107 to CD11bA did not trigger the activating tertiary changes in the domain or in the full-length integrin. These data show that the denticity of the ligand Asp/Glu can modify the divalent cation selectivity at MIDAS and hence integrin function. Stabilizing the Ca2+ ion at MIDAS by bidentate ligation to a ligand Asp/Glu may provide one approach for designing pure integrin antagonists.

Mutations in optineurin have recently been linked to amyotrophic lateral sclerosis (ALS).To determine whether optineurin-positive skeinlike inclusions are a common pathologic feature in ALS, including SOD1 -linked ALS.Clinical case series.Academic referral center.We analyzed spinal cord sections from 46 clinically and pathologically diagnosed ALS cases and ALS transgenic mouse models overexpressing ALS-linked SOD1 mutations G93A or L126Z.We observed optineurin-immunoreactive skeinlike inclusions in all the sporadic ALS and familial ALS cases without SOD1 mutation, but not in cases with SOD1 mutations or in transgenic mice overexpressing the ALS-linked SOD1 mutations G93A or L126Z.The data from this study provide evidence that optineurin is involved in the pathogenesis of sporadic ALS and non- SOD1 familial ALS, thus supporting the hypothesis that these forms of ALS share a pathway that is distinct from that of SOD1-linked ALS.

Amyotrophic lateral sclerosis (ALS) is a paralytic and usually fatal disorder caused by motor-neuron degeneration in the brain and spinal cord. Most cases of ALS are sporadic but about 5-10% are familial. Mutations in superoxide dismutase 1 (SOD1), TAR DNA-binding protein (TARDBP, also known as TDP43) and fused in sarcoma (FUS, also known as translocated in liposarcoma (TLS)) account for approximately 30% of classic familial ALS. Mutations in several other genes have also been reported as rare causes of ALS or ALS-like syndromes. The causes of the remaining cases of familial ALS and of the vast majority of sporadic ALS are unknown. Despite extensive studies of previously identified ALS-causing genes, the pathogenic mechanism underlying motor-neuron degeneration in ALS remains largely obscure. Dementia, usually of the frontotemporal lobar type, may occur in some ALS cases. It is unclear whether ALS and dementia share common aetiology and pathogenesis in ALS/dementia. Here we show that mutations in UBQLN2, which encodes the ubiquitin-like protein ubiquilin 2, cause dominantly inherited, chromosome-X-linked ALS and ALS/dementia. We describe novel ubiquilin 2 pathology in the spinal cords of ALS cases and in the brains of ALS/dementia cases with or without UBQLN2 mutations. Ubiquilin 2 is a member of the ubiquilin family, which regulates the degradation of ubiquitinated proteins. Functional analysis showed that mutations in UBQLN2 lead to an impairment of protein degradation. Therefore, our findings link abnormalities in ubiquilin 2 to defects in the protein degradation pathway, abnormal protein aggregation and neurodegeneration, indicating a common pathogenic mechanism that can be exploited for therapeutic intervention.

Objective: To report a novel causative gene for an autosomal dominant mitochondrial myopathy. Background Mitochondrial myopathies belong to a larger group of systemic diseases caused by morphological or biochemical abnormalities of mitochondria. Mitochondrial disorders can be caused by mutation either in the mitochondrial or in the nuclear genome. Design/Methods: Patients are part of previously reported Hispanic kindred with autosomal dominant mitochondrial myopathy (Heiman-Patterson et al 1997). Linkage analysis was established using fluorescent microsatellite markers and illumina genome wide SNP markers. Candidate gene was identified using MitoCarta (Vamsi K. Mootha, personal communication). Candidate gene was sequenced using Beckman Coulter CEQ 8000 DNA analysis system. The cDNA of wild type, single and double mutant gene were cloned in pCMV-Myc vector and co-transfected with pDsRed2-mito in HEK293 cells. The co-localization was monitored with confocal microscopy. Results: In this five generation Hispanic family we established linkage to chromosome 22q11. Using the tools of integrative gneomics, we established CHCHD10/ C22orf16 as the high scoring mitochondrial candidate gene in our minimal candidate region. Sequence analysis revealed a double heterozygote missense mutation in this gene. These two mutations segregated with disease phenotype and were absent in 500 Caucasians and Hispanic non affected controls. Both wild types, single and double mutant proteins co-localized with mitochondria. Conclusions: We identified a new nuclear encoded mitochondrial gene ( CHCHD10 ) as the cause of an autosomal dominant mitochondrial myopathy. CHCHD10 encodes a non characterized 142 aa protein with a target mitochondrial signaling peptide and a conserved CHCH domain. This CHCH domain is present in many intermembrane space proteins, such as the family of small Tim proteins. The exact function of this protein remains to be determined. Our findings expands the spectrum of mitochondrial myopathies caused by nuclear gene dysfunction. Disclosure: Dr. Ajroud-Driss has nothing to disclose. Dr. Fecto has nothing to disclose. Dr. Ajroud has nothing to disclose. Dr. Siddique has nothing to disclose.

Objective: To identify the genetic defect causing dominant X-linked ALS and ALS/dementia, and characterize the functional and pathological determinants. Background Most cases of ALS are sporadic but about 5-10% are familial. Mutations in SOD1, TDP43 and FUS account for approximately 30% of classic familial ALS. The causes of the remaining cases of familial ALS and of the vast majority of sporadic ALS are unknown. Despite extensive studies of previously identified ALS-causing genes, the pathogenic mechanism underlying motor-neuron degeneration in ALS remains largely obscure. Dementia, usually of the frontotemporal lobar type, may occur in some ALS cases. It is unclear whether ALS and dementia share common aetiology and pathogenesis in ALS/dementia. More than a decade ago, we had linked a large ALS family to the pericentric region of the X chromosome. Design/Methods: Detailed mapping was done with dense microsatellite markers and Illumina Sentrix HumanHap300 Genotyping BeadChip. Candidate genes were sequenced using Beckman Coulter CEQ 8000 DNA analysis system. Immunohistochemistry and light and confocal microscopy were performed according to standard protocols. Neuro2a and SHSY-5Y cells were transiently co-transfected with a ubiquitin-proteasome system (UPS) reporter plasmid (Ub-G76V-GFP) and pIRES2-DsRed2 dual expression vectors containing either wildtype or mutant UBQLN2 and analyzed using a MoFlo cell sorter and Summit software. Results: We show mutations of ubiquilin2, a ubiquitin-like protein, in five families with ALS and ALS/dementia. We also show that inclusions containing ubiquilin2 are a common pathological feature in a wide spectrum of ALS and ALS/dementia. Functional studies indicate an impairment of ubiquitin-mediated proteasomal degradation in cells expressing mutant ubiquilin2. Conclusions: These data provide evidence for an impairment of protein turnover in the pathogenesis of ALS and ALS/dementia. Further elucidation of these processes may be central to the understanding of pathogenic pathways. These pathways should provide novel molecular targets for the design of rational therapies for these disorders. Supported by: The National Institute of Neurological Disorders and Stroke (NS050641), the Les Turner ALS Foundation, the Vena E. Schaff ALS Research Fund, the Harold Post Research Professorship, the Herbert and Florence C. Wenske Foundation, the David C. Asselin MD Memorial Fund, the Help America Foundation and the Les Turner ALS Foundation/Herbert C. Wenske Foundation Professorship. F.F. has support from NIH (T32 AG20506). K.A. is a postdoctoral fellow of the Blazeman Foundation for ALS. G.H.G. received travel funds from MND Scotland. Disclosure: Dr. Fecto has nothing to disclose. Dr. Deng has nothing to disclose. Dr. Chen has nothing to disclose. Dr. Hong has nothing to disclose. Dr. Boycott has nothing to disclose. Dr. Gorrie has nothing to disclose. Dr. Siddique has nothing to disclose. Dr. Yang has nothing to disclose. Dr. Shi has nothing to disclose. Dr. Zhai has nothing to disclose. Dr. Jiang has nothing to disclose. Dr. Hirano has nothing to disclose. Dr. Rampersaud has nothing to disclose. Dr. Jansen has nothing to disclose. Dr. Donkervoort has nothing to disclose. Dr. Bigio has nothing to disclose. Dr. Brooks has received personal compensation for activities with Avanir Pharmaceuticals, Bayer Healthcare Pharmaceuticals, Biogen Idec, Genentech, Inc., and Teva Neuroscience. Dr. Brooks has received research support from Avanir Pharmaceuticals, Biogen Idec, NINDS, Novartis, and Teva Neuroscience. Dr. Ajroud has nothing to disclose. Dr. Sufit has nothing to disclose. Dr. Haines has nothing to disclose. Dr. Mugnaini has nothing to disclose. Dr. Pericak-Vance has nothing to disclose. Dr. Siddique has nothing to disclose.

Background Mutations in optineurin have recently been linked to amyotrophic lateral sclerosis (ALS). Objective To determine whether optineurin-positive skeinlike inclusions are a common pathologic feature in ALS, including SOD1 -linked ALS. Design Clinical case series. Setting Academic referral center. Subjects We analyzed spinal cord sections from 46 clinically and pathologically diagnosed ALS cases and ALS transgenic mouse models overexpressing ALS-linked SOD1 mutations G93A or L126Z. Results We observed optineurin-immunoreactive skeinlike inclusions in all the sporadic ALS and familial ALS cases without SOD1 mutation, but not in cases with SOD1 mutations or in transgenic mice overexpressing the ALS-linked SOD1 mutations G93A or L126Z. Conclusion The data from this study provide evidence that optineurin is involved in the pathogenesis of sporadic ALS and non- SOD1 familial ALS, thus supporting the hypothesis that these forms of ALS share a pathway that is distinct from that of SOD1 -linked ALS.